Abstract

Adenovirus was first identified in the 1950s and since then this pathogenic group of viruses has been explored and transformed into a genetic transfer vehicle. Modification or deletion of few genes are necessary to transform it into a conditionally or non-replicative vector, creating a versatile tool capable of transducing different tissues and inducing high levels of transgene expression. In the early years of vector development, the application in monogenic diseases faced several hurdles, including short-term gene expression and even a fatality. On the other hand, an adenoviral delivery strategy for treatment of cancer was the first approved gene therapy product. There is an increasing interest in expressing transgenes with therapeutic potential targeting the cancer hallmarks, inhibiting metastasis, inducing cancer cell death or modulating the immune system to attack the tumor cells. Replicative adenovirus as vaccines may be even older and date to a few years of its discovery, application of non-replicative adenovirus for vaccination against different microorganisms has been investigated, but only recently, it demonstrated its full potential being one of the leading vaccination tools for COVID-19. This is not a new vector nor a new technology, but the result of decades of careful and intense work in this field.

Keywords:
Adenovirus; gene therapy; monogenic diseases; cancer; vaccines

Introduction

Adenoviruses were first identified in 1953 after an analysis of tissue culture of tonsils and adenoids, that was aiming to identify unknown viruses from the respiratory tract that could cause acute respiratory diseases. Huebner et al. identified 13 new agents in surgically removed adenoids. Because the main symptoms presented by patients were acute pharyngitis and conjunctivitis, the authors proposed the term “adenoidal-pharyngeal-conjunctival agents” to designate this group of viruses, but posteriorly the name has changed to adenovirus, referring to the tissue of its first reported isolation (Robbins et al., 1950Robbins FC, Enders JF and Weller TH (1950) Cytopathogenic effect of poliomyelitis viruses in vitro on, human embryonic tissues. Proc Soc Exp Biol Med 75:370-374. ; Huebner et al., 1954Huebner RJ, Rowe WP, Ward TG, Parrott RH and Bell JA (1954) Adenoidal-pharyngeal-conjunctival agents: a newly recognized group of common viruses of the respiratory system. N Engl J Med 251:1077-1086. ).

Data provided from the Journal of Gene Medicine indicates that adenoviral vectors are the most used vector type for gene transfer, representing 17.5% of all gene therapy clinical trials (Gene Therapy Clinical Trials Worldwide - GTCT, 2021Gene Therapy Clinical Trials Worldwide (GTCT), Gene Therapy Clinical Trials Worldwide (GTCT), http://www.abedia.com/wiley/index.html (accessed 27 February 2022).
http://www.abedia.com/wiley/index.html... ). They are most commonly employed in cancer therapies, but can also be applied in vaccinal approaches and treatment of monogenic diseases. Its extensive applications are due to intrinsic adenoviral vector characteristics: non-integration in the host genome and high capacity for gene transfer and storage. Although adenoviruses are pathogenic and associated with respiratory and gastrointestinal diseases, modifications of their genome have been made to turn the adenoviral vectors safe and to avoid adverse effects of the therapy. These genetic modifications on the viral genome generated a replication-defective vector, preventing a high viral load in the host body. The evolution of adenoviral vectors development is shown in Figure 1.

First generation adenoviral vectors

In 1977, a cell line that is necessary for recombinant non-replicative adenoviral vectors was raised. The human embryonic kidney (HEK) cells were modified with human adenovirus type 5 (Ad5) DNA fragments and the particular clone 293 (HEK293) was transformed by the acquisition of 4 copies of the left end of Ad5 genome, a region that includes the E1 gene. Thus, HEK293 was the first established human cell transformed by an adenovirus (Graham et al., 1977Graham FL, Smiley J, Russell WC and Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal Gen Virol 36:59-72. ), which made possible the development of the first generation of recombinant adenoviruses presenting deletions in the E1 and E3 genes, that are associated with the expression of all other genes involved in viral replication and inhibition of host immune system, respectively.

The E1 region is divided into two parts: E1a and E1b. A group of mutants with deletions on region E1 was isolated and infected HEK293 cells in vitro to observe if adenoviruses were able of growing on it (Jones and Shenk, 1978Jones N and Shenk T (1978) Isolation of deletion and substitution mutants of adenovirus type 5. Cell 13:181-188. , 1979aJones N and Shenk T (1979a) An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A 76:3665-3669. ,bJones N and Shenk T (1979b) Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell 17:683-689. ). The authors identified two mutants, one lacking E1a (deletion of 902 bp, around position 540-1620 bp of the genome) and other E1b (deletion of 2350 pb, around position 1260-3780 bp of the genome) that were able to replicate in HEK293 cell lines, but neither in HeLa nor HEK cells. Concluding that E1 gene was necessary for viral growth, which was only possible in HEK293 cells, that contains the E1 gene supporting viral replication of the mutant adenoviruses. Later, deletions with a maximum of 3 kb have been made in this region to generate E1 deleted adenovirus vectors with a capacity of insertion up to 5 kb (Rosenfeld et al., 1991Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Pääkkö PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M et al. (1991) Adenovirus-mediated transfer of a recombinant α1-Antitrypsin Gene to the Lung Epithelium in vivo. Science 252:431-434. ).

The E3 gene has its expression activated by E1a gene product and encodes proteins that counteract the attack of the immune system and prevents programmed cell death. Thus, the E3 gene products are not related to viral replication, and therefore no complementing cell line is necessary (Wold et al., 1995Wold WSM, Tollefson AE and Hermiston TW (1995) E3 transcription unit of adenovirus. Curr Top Microbiol Immunol 199:237-274. ). Viral vector with deletion of the E3 gene (from 28kb to 30kb) was indistinguishable from wild type (WT) adenovirus in growth kinetics (Cladaras et al., 1985Cladaras C, Bhat B and Wold WSM (1985) Mapping the 5’ ends, 3’ ends, and splice sites of mRNAs from the early E3 transcription unit of adenovirus 5. Virology 140:44-54. ). Vectors deleted in the E1 and E3 genes have a storage capacity of approximately 8 kb.

Second-generation adenoviral vectors

Although adenoviral vectors are successful in gene transferring and expression, some concerns were raised. In vivo delivery of recombinant adenoviral vector carrying the LacZ gene in the liver showed low levels of transgene expression and induction of cellular immune response, leading to destruction of genetically modified hepatocytes and repopulation with parental cells, without the transgene (Yang et al., 1994Yang Y, Nunes FA, Berencsi K, Furth EE, Gönczöl E and Wilson JM (1994) Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A 91:4407). This probably occurred due to the background expression and accumulation of viral late genes, leading to inflammation and destruction of transduced cells (Gilgenkrantz et al., 1995Gilgenkrantz H, Duboc D, Juillard V, Couton D, Pavirani A, Guillet JG, Briand P and Kahn A (1995) Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response. Hum Gene Ther 6:1265-1274. ; Yang et al., 1996Yang Y, Su Q and Wilson JM (1996) Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J Virol 70:7209-7212. ). In view of that, new recombinant adenovirus needed to be developed, with new mutations on other viral genes. In this context, second generation adenoviral vectors were developed, including additional mutations or deletions of E2 and E4 genes. Both genes participate in the expression of late genes, and their absence reduced adverse effects caused by the expression of the late genes. Furthermore, there was an increased storage capacity, allowing it to accommodate up to 14 kb.

In 1994, the first modification of first-generation adenovirus was performed. Alongside E1 deletion, it incorporated a mutation into the E2a region, turning this gene temperature-sensitive (Engelhardt et al., 1994Engelhardt JF, Ye X, Doranz B and Wilson JM (1994) Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci U S A 91:6196-6200. ). E2a gene encodes a single-stranded DNA binding protein that is responsible for DNA synthesis. Recombinant adenovirus with E2a temperature-sensitive mutation has reduced late protein expression levels. In contrast, the E4 gene encodes two ORFs, ORF3 and ORF6, which participate in viral DNA synthesis and expression of late genes (Sandler and Ketner, 1989Sandler AB and Ketner G (1989) Adenovirus early region 4 is essential for normal stability of late nuclear RNAs. J Virol 63:624. ). ORF 6 gene product forms a complex with E1b and mediates the transport of viral messenger RNA from the nucleus to the cytoplasm and ORF 3 gene product acts in parallel with this complex to enable viral DNA replication. Deletion of the E4 gene blocks adenoviral replication and is lethal (Halbert et al., 1985Halbert DN, Cutt JR and Shenk T (1985) Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J Virol 56:250. ). Differently of E2 temperature-sensitive mutation, deletion of the E4 gene entails the need of a cell line capable of complementing its absence, but without overexpressing cytotoxic late proteins. In 1995, a HEK293 cell line expressing E4 was established by introducing a full-length E4 region under control of the mouse alpha inhibin promoter, enabling the production of E1/E4-deleted adenovirus vectors (Wang et al., 1995Wang Q, Jia XC and Finer HM (1995) A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene-region deletions. Semantic Scholar. Gene Ther 775-783.). However, even with these new gene deletions, second-generation vectors still do not avoid completely in vivo immunogenicity (Lusky et al., 1998Lusky M, Christ M, Rittner K, Dieterle A, Dreyer D, Mourot B, Schultz H, Stoeckel F, Pavirani A and Mehtali MJ (1998) In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted. Virology 72:2022-2032. ).

Different barriers have been considered for safe and effective adenoviral-mediated gene therapy such as: (1) the severe innate and adaptative immune responses against vectors and transgenes that lead to severe adverse side effects (Tripathy et al., 1996Tripathy SK, Black HB, Goldwasser E and Leiden JM (1996) Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovims vectors. Nat Med 2:545-50.; Harvey et al., 2002Harvey BG, McKinney RL, Rosengart T, Lesser ML and Crystal RG (2002) Systemic interleukin-6 responses following administration of adenovirus gene transfer vectors to humans by different routes. Mol Ther 6:287-297.); (2) the high pre-existing immunity against adenovirus in the population (Barouch et al., 2011Barouch DH, Kik SV, Weverling GJ, Dilan R, King SL, Maxfield LF, Clark S, Ng’ang’a D, Brandariz KL, Abbink P et al. (2011) International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations. Vaccine 29:5203-5209. ; Ye et al., 2018Ye X, Xiao L, Zheng X, Wang J, Shu T, Feng Y, Liu X, Su W, Wang Q, Li C et al. (2018) Seroprevalence of neutralizing antibodies to human adenovirus type 4 and 7 in healthy populations from southern China. Front Microbiol 9:3040. ) that can hamper the efficacy of the treatment due to neutralizing antibodies that rapidly blocks the virus (Tripathy et al., 1996Tripathy SK, Black HB, Goldwasser E and Leiden JM (1996) Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovims vectors. Nat Med 2:545-50.; Kushwah et al., 2008Kushwah R, Cao H and Hu J (2008) Characterization of Pulmonary T Cell Response to Helper-Dependent Adenoviral Vectors following Intranasal Delivery. J Immunol 180:4098-4108.; Parker et al., 2009Parker AL, Waddington SN, Buckley SMK, Custers J, Havenga MJE, van Rooijen N, Goudsmit J, McVey JH, Nicklin SA and Baker AH (2009) Effect of Neutralizing Sera on Factor X-Mediated Adenovirus Serotype 5 Gene Transfer. J Virol 83:479-483. ); (3) the elimination of Ad vectors through liver and spleen after intravenous applications due to interactions between Ad vector and host proteins (Parker et al., 2006Parker AL, Waddington SN, Nicol CG, Shayakhmetov DM, Buckley SM, Denby L, Kemball-Cook G, Ni S, Lieber A, McVey JH et al. (2006) Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes. Blood 108:2554-61. ); (4) the natural tropism of most adenovirus through the attachment of the Ad fiber knob protein with CAR, which is expressed in a huge range of tissues making it difficult to transduce only specific cells (Bewley et al., 1999Bewley MC, Springer K, Zhang YB, Freimuth P and Flanagan JM (1999) Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR. Science 286:1579-15783. ; Einfeld et al., 2001Einfeld DA, Schroeder R, Roelvink PW, Lizonova A, King CR, Kovesdi I and Wickham TJ (2001) Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions. J Virol 75:11284-11291. ). Therefore, additional alterations in the adenoviral vectors have been developed.

Third-generation adenoviral vectors

To resolve some of these questions, the third and last generation of adenoviral vectors were created, also called gutless, helper-dependent (HD-Ad), or high-capacity (HC-Ad) vectors. This vector has all the viral genes deleted, keeping only the ITRs and the packaging sequence. Because of this modification, the HC-Ad vector needs a helper adenoviral vector encoding all viral genes. When both vectors are coinfected in a eukaryotic cell line, the helper adenovirus produces the structural proteins, which will be assembled into the capsid particle incorporating the HC-Ad genome. Due to the deletion of all viral genes, the helper-dependent adenovirus has a capacity of gene insertion up to 37 kb. The biggest limitation for its broader use is the incorporation of the helper virus genome into the capsid. Therefore, the final product is a mixture of HC-Ad and contaminating helper virus (Alba et al., 2005Alba R, Bosch A, Chillon M. Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther 2005 12:S18-27. ). The first strategy that tried to overcome this problem was developed by Mitani and colleaagues who used an Ad5 with a defective packaging signal as the helper virus, while the gutless vector had deletions of only L1, L2, VA, and TP genes. However, during viral vector production, both HC-Ad and helper virus were obtained (Mitani et al., 1995Mitani K, Graham FL, Caskey CT and Kochanek S (1995) Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci U S A 92:3854-3858. ).

An important advance was the development of Ad helper virus containing the packaging signals flanked byloxP sites, which were excised by the Cre recombinase, rendering the helper virus genome unpackageable and producing high titers of the vector with very low quantities of contaminating helper virus, which was still present at a range around 0,1% - 10% (Parks et al., 1996Parks RJ, Chen L, Anton M, Sankar U, Rudnicki MA and Graham FL (1996) A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A 93:13565. ). These high levels of contamination were due to the enzyme activity, that cannot remove 100% of packaging signals in the helper virus. Indeed, this system provides increased cloning capacity, safety, and reduced immunogenicity, but contamination by helper virus is still a problem.

Since the development of this system, many similar techniques have been developed and they suffer from the same problems: the difficulty of vector production and the presence of helper virus contaminations. Another improvement in Cre/loxP system was based on the reversion of the packaging sequence of helper adenovirus. This system provided lower levels of helper contaminations, around 0,02 - 0,1%, and improved vector production (Palmer and Ng, 2003Palmer D and Ng P (2003) Improved system for helper-dependent adenoviral vector production. Mol Ther 8:846-852. ). Other recombinase systems were also explored, such as the FLP/frt system (Ng et al., 2001Ng P, Beauchamp C, Evelegh C, Parks R and Graham FL (2001) Development of a FLP/frt system for generating helper-dependent adenoviral vectors. Mol Ther 3:809-815. ) and the Vika recombinase system (Phillips et al., 2022Phillips S, Ramos PV, Veeraraghavan P and Young SM (2022) VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production. Mol Ther Methods Clin Dev 24:117-126. ). However, none of these approaches completely eliminates the presence of the helper virus.

The Helper-virus-free strategy involves the co-transfection of the HC-Ad with a helper plasmid. Using this approach, vectors expressing the human dystrophin and huntingtin genes were produced on large scale and efficiently delivered into cells and mouse models, showing therapeutic potential for Huntington’s disease and Duchenne muscular dystrophy (Lee et al., 2019Lee D, Liu J, Junn HJ, Lee E-JJ, Jeong K-SS and Seol D-WW (2019) No more helper adenovirus: production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants. Exp Mol Med 51:1-18. ).

Comparing replicative, first-generation, and HC-Ad for vaccination purposes, it was observed that replicative and HC-Ad induced stronger humoral immune response, but not cellular immune response, while HC-Ad also induced lower ALT levels compared with replicative and first-generation adenoviral vectors, indicating a possible reduced liver toxicity (Weaver et al., 2009Weaver EA, Nehete PN, Buchl SS, Senac JS, Palmer D, Ng P, Sastry KJ and Barry MA (2009) Comparison of replication-competent, first generation, and helper-dependent adenoviral vaccines. PloS One 4:e5059. ).

Conditionally replicative adenoviral vectors

Besides all attempts and modifications involving replicative-defective adenovirus, a different approach maintains its replication capacity. Conditionally replicating adenoviruses have been employed as oncolytic adenoviruses, showing replicative potential only in tumor cells, destroying them in the process and continuously disseminating and replicating in cancer cells. One of the first examples is the Onyx-015, which has an alteration in the E1B-55K gene (Bischoff et al., 1996Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A et al. (1996) An Adenovirus Mutant That Replicates Selectively in p53- Deficient Human Tumor Cells. Science 274:373-376. ). Lack of E1B-55K inhibited late viral RNA export from the nucleus to the cytoplasm preventing expression of late genes in normal cells. However, in tumor cells, the viral RNA is exported independently of the presence of E1B-55K and viral proteins expression and replication occurs (O’Shea et al., 2004O’Shea CC, Johnson L, Bagus B, Choi S, Nicholas C, Shen A, Boyle L, Pandey K, Soria C, Kunich J et al. (2004) Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell 6:611-623. ). Oncorine (Creative Biolabs, Inc., Shirley, NY) is similar to ONYX-015 and was the first oncolytic adenovirus approved for the treatment of nasopharyngeal carcinoma in China (Liang, 2018Liang M (2018) oncorine, the world first oncolytic virus medicine and its update in China. Curr Cancer Drug Targets 18:171-176. ). Further examples are seen in Adenovirus in cancer gene therapy section.

Investigation of other adenovirus types and modifications

All early adenoviral studies were conducted in type 2 and 5 human adenoviruses, therefore gathered knowledge is deeper in these types compared to other adenoviruses. However, the presence of neutralizing antibodies (Dudareva et al., 2009Dudareva M, Andrews L, Gilbert SC, Bejon P, Marsh K, Mwacharo J, Kai O, Nicosia A and Hill AVS (2009) Prevalence of serum neutralizing antibodies against chimpanzee adenovirus 63 and human adenovirus 5 in Kenyan children, in the context of vaccine vector efficacy. Vaccine 27:3501-3504. ; Pilankatta et al., 2010Pilankatta R, Chawla T, Khanna N and Swaminathan S (2010) The prevalence of antibodies to adenovirus serotype 5 in an adult Indian population and implications for adenovirus vector vaccines. J Med Virol 82:407-414. ; Barouch et al., 2011Barouch DH, Kik SV, Weverling GJ, Dilan R, King SL, Maxfield LF, Clark S, Ng’ang’a D, Brandariz KL, Abbink P et al. (2011) International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations. Vaccine 29:5203-5209. ; Zhang et al., 2013bZhang S, Huang W, Zhou X, Zhao Q, Wang Q and Jia B (2013b) Seroprevalence of neutralizing antibodies to human adenoviruses type-5 and type-26 and chimpanzee adenovirus type-68 in healthy Chinese adults. J Med Virol 85:1077-1084. ; Su et al., 2016Su X, Tian X, Jiang Z, Ma Q, Liu Q, Lu X and Zhou R (2016) Human Adenovirus Serotype 3 Vector Packaged by a Rare Serotype 14 Hexon. PloS One 11:e0156984. ; Zhao et al., 2018Zhao H, Xu C, Luo X, Wei F, Wang N, Shi H and Ren X (2018) Seroprevalence of neutralizing antibodies against human adenovirus type-5 and chimpanzee adenovirus type-68 in cancer patients. Front Immunol 9:335. ) may impair gene transfer mediated by them. Cotton rats previously infected with WT Ad5 had reduced immunization efficacy mediated by an Ad5 non-replicative vector (Papp et al., 1999aPapp Z, Babiuk LA and Baca-Estrada ME (1999a) The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1. Vaccine 17:933-943. ).

In order to overcome this problem, other adenoviruses have been evaluated. Ad35 has a low global prevalence and has been further studied (Gao et al., 2003Gao W, Robbins PD and Gambotto A (2003) Human adenovirus type 35: nucleotide sequence and vector development. Gene Ther 10:1941-1949. ; Vogels et al., 2003Vogels R, Zuijdgeest D, Rijnsoever R van, Hartkoorn E, Damen I, Béthune M-P, Kostense S, Penders G, Helmus N, Koudstaal W et al. (2003) Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity. J Virol 77:8263. ; Nwanegbo et al., 2004Nwanegbo E, Vardas E, Gao W, Whittle H, Sun H, Rowe D, Robbins PD and Gambotto A (2004) Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States. Clin Diagn Lab Immunol 11:351-357. ). It has a tropism to cells with CD46 receptor rather than cells expressing CAR (coxsackie and adenovirus receptor), but this can be overcome by construction of a chimeric Ad35 expressing the Ad5 fiber knob (Nanda et al., 2005Nanda A, Lynch DM, Goudsmit J, Lemckert AAC, Ewald BA, Sumida SM, Truitt DM, Abbink P, Kishko MG, Gorgone DA et al. (2005) Immunogenicity of Recombinant Fiber-Chimeric Adenovirus Serotype 35 Vector-Based Vaccines in Mice and Rhesus Monkeys. J Virol 79:14161-14168. ). Several other adenoviruses with low seroprevalence have been engineered into non-replicative adenoviral vectors, such as: Ad11 (Holterman et al., 2004Holterman L, Vogels R, van der Vlugt R, Sieuwerts M, Grimbergen J, Kaspers J, Geelen E, van der Helm E, Lemckert A, Gillissen G et al. (2004) Novel Replication-Incompetent Vector Derived from Adenovirus Type 11 (Ad11) for Vaccination and Gene Therapy: Low Seroprevalence and Non-Cross-Reactivity with Ad5. J Virol 78:13207-13215. ); Ad41 (Lemiale et al., 2007Lemiale F, Haddada H, Nabel GJ, Brough DE, King CR and Gall JGD (2007) Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41. Vaccine 25:2074. ); Ad56 (Duffy et al., 2018Duffy MR, Alonso-Padilla J, John L, Chandra N, Khan S, Ballmann MZ, Lipiec A, Heemskerk E, Custers J, Arnberg N et al. (2018) Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 56. J Gen Virol 99:135-147. ); Ad19a, which transduces dendritic cells (Ragonnaud et al., 2018Ragonnaud E, Schroedel S, Mariya S, Iskandriati D, Pamungkas J, Fougeroux C, Daradoumis J, Thomsen AR, Neukirch L, Ruzsics Z et al. (2018) Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques. Vaccine 36:6212-6222. ); Ad20-42-42, which is related to type 42 but with a penton base derived from type 20 and tropism to both CAR and CD46 receptors (Ballmann et al., 2021Ballmann MZ, Raus S, Engelhart R, Kaján GL, Beqqali A, Hadoke PWF, van der Zalm C, Papp T, John L, Khan S et al. (2021) Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development. J Virol 95:e0038721. ); Ad26, Ad48 and Ad50 are rare types, the Ad26 vector was shown to be the most immunogenic and more interesting in vaccine development (Abbink et al., 2007Abbink P, Lemckert AAC, Ewald BA, Lynch DM, Denholtz M, Smits S, Holterman L, Damen I, Vogels R, Thorner AR et al. (2007) Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J Virol 81:4654-4663. ). Ad26 uses sialic acid as a primary target in the cell (Baker et al., 2019Baker AT, Mundy RM, Davies JA, Rizkallah PJ and Parker AL (2019) Human adenovirus type 26 uses sialic acid-bearing glycans as a primary cell entry receptor. Sci Adv 5:eaax3567. ).

The Ad5 can be altered to reduce the binding of neutralizing antibodies. The adenoviral hexon protein is a major component that drives the host immune response. Replacing the hexon of Ad5 with the one from Ad3 reduced neutralization of viral particles (Yan et al., 2021Yan Y, Jing S, Feng L, Zhang J, Zeng Z, Li M, Zhao S, Ou J, Lan W, Guan W et al. (2021) Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine: “Adenovirus Vaccine Within an Adenovirus Vector”. Virol Sin 36:354-364. ). As well exchange of the hexon gene of Ad3 with the hexon from Ad14 generated a chimeric vector that was not neutralized by antibodies against Ad3 (Su et al., 2016Su X, Tian X, Jiang Z, Ma Q, Liu Q, Lu X and Zhou R (2016) Human Adenovirus Serotype 3 Vector Packaged by a Rare Serotype 14 Hexon. PloS One 11:e0156984. ). Modifications of hypervariable regions within the hexon gene could also impair antibodies against Ad5 binding. A chimeric hexon protein from Ad5, with replacement of some regions from Ad48, circumvented pre-existing immunogenicity (Roberts et al., 2006Roberts DM, Nanda A, Havenga MJE, Abbink P, Lynch DM, Ewald BA, Liu J, Thorner AR, Swanson PE, Gorgone DA et al. (2006) Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 441:239-243. ; Teigler et al., 2014Teigler JE, Penaloza-MacMaster P, Obeng R, Provine NM, Larocca RA, Borducchi EN and Barouch DH (2014) Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5) Vectors Display Reduced Hepatotoxicity but Induce T Lymphocyte Phenotypes Similar to Ad5 Vectors. Clin Vaccine Immunol 21:1137-1444. ). Modification of a hypervariable region 2 of Ad5 with the region from Ad3 also reduced neutralization (Gu et al., 2016Gu L, Icyuz M, Krendelchtchikova V, Krendelchtchikov A, Johnston AE and Matthews QL (2016) Development of an Ad5H3 Chimera Using the “Antigen Capsid-Incorporation” Strategy for an Alternative Vaccination Approach. Open Virol J 10:10-20. ). Alteration of all hypervariable regions from Ad5 introducing the regions from Ad43 had the same effect (Bruder et al., 2012Bruder JT, Semenova E, Chen P, Limbach K, Patterson NB, Stefaniak ME, Konovalova S, Thomas C, Hamilton M, King CR et al. (2012) Modification of Ad5 hexon hypervariable regions circumvents pre-existing Ad5 neutralizing antibodies and induces protective immune responses. PloS One 7:e33920. ). Epitope modification in the 5^th hypervariable region of Ad5 also prevented antibody neutralization (Abe et al., 2009Abe S, Okuda K, Ura T, Kondo A, Yoshida A, Yoshizaki S, Mizuguchi H, Klinman D and Shimada M (2009) Adenovirus type 5 with modified hexons induces robust transgene-specific immune responses in mice with pre-existing immunity against adenovirus type 5. J Gene Med 11:570-579. ). Modification of both hexon and fiber proteins abrogated adenoviral vector neutralization (Bradley et al., 2012Bradley RR, Lynch DM, Iampietro MJ, Borducchi EN and Barouch DH (2012) Adenovirus Serotype 5 Neutralizing Antibodies Target both Hexon and Fiber following Vaccination and Natural Infection. J Virol 86:625. ), and chimeric Ad5 with fiber from Ad35 escaped neutralization (Flickinger et al., 2020Flickinger JC, Singh J, Carlson R, Leong E, Baybutt TR, Barton J, Caparosa E, Pattison A, Rappaport JA, Roh J et al. (2020) Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity. J Immunother Cancer 8:e001046. ).

Additionally, several adenoviruses infecting other mammals and capable of infecting human cells have been investigated, including adenovirus from bovine type 3 (Mittal et al., 1995Mittal SK, Prevec L, Graham FL and Babiuk LA (1995) Development of a bovine adenovirus type 3-based expression vector. J Gen Virol 76:93-102. ), chimpanzee (ChAd) type 68 (Xiang et al., 2002Xiang Z, Gao G, Reyes-Sandoval A, Cohen CJ, Li Y, Bergelson JM, Wilson JM and Ertl HCJ (2002) Novel, Chimpanzee Serotype 68-Based Adenoviral Vaccine Carrier for Induction of Antibodies to a Transgene Product. J Virol 76:2667-2675. ), types 5, 6, 7 (Roy et al., 2004Roy S, Gao G, Lu Y, Zhou X, Lock M, Calcedo R and Wilson JM (2004) Characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors. Hum Gene Ther 15:519-530. ), C1 (Tatsis et al., 2007aTatsis N, Blejer A, Lasaro MO, Hensley SE, Cun A, Tesema L, Li Y, Gao GP, Xiang ZQ, Zhou D et al. (2007a) A CD46-binding Chimpanzee Adenovirus Vector as a Vaccine Carrier. Mol Ther 15:608-617. ) and Y25 (Dicks et al., 2012Dicks MDJ, Spencer AJ, Edwards NJ, Wadell G, Bojang K, Gilbert SC, Hill AVS and Cottingham MG (2012) A Novel Chimpanzee Adenovirus Vector with Low Human Seroprevalence: Improved Systems for Vector Derivation and Comparative Immunogenicity. PloS One 7:40385. ), rhesus monkey types 51, 52 and 53 (Abbink et al., 2015Abbink P, Maxfield LF, Ng’ang’a D, Borducchi EN, Iampietro MJ, Bricault CA, Teigler JE, Blackmore S, Parenteau L, Wagh K et al. (2015) Construction and evaluation of novel rhesus monkey adenovirus vaccine vectors. J Virol 89:1512-1522. ), porcine type 3 (Bangari and Mittal, 2004Bangari DS and Mittal SK (2004) Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture. Virus Res 105:127-136. ) and simian type 21 (Roy et al., 2006Roy S, Zhi Y, Kobinger GP, Figueredo J, Calcedo R, Miller JR, Feldmann H and Wilson JM (2006) Generation of an adenoviral vaccine vector based on simian adenovirus 21. J Gen Virol 87:2477-2485. ). Clinical trial data indicated that ChAd63 is safe and induces a strong immune response (O’Hara et al., 2012O’Hara GA, Duncan CJA, Ewer KJ, Collins KA, Elias SC, Halstead FD, Goodman AL, Edwards NJ, Reyes-Sandoval A, Bird P et al. (2012) Clinical Assessment of a Recombinant Simian Adenovirus ChAd63: A Potent New Vaccine Vector. J Infect Dis 205:772-781. ). A vector derived from ChAdY25 was obtained by removal of E1 and E3 genes and the E4 gene was modified to optimize growth rate in human cell lines, generating the ChAdOx1(Dicks et al., 2012Dicks MDJ, Spencer AJ, Edwards NJ, Wadell G, Bojang K, Gilbert SC, Hill AVS and Cottingham MG (2012) A Novel Chimpanzee Adenovirus Vector with Low Human Seroprevalence: Improved Systems for Vector Derivation and Comparative Immunogenicity. PloS One 7:40385. ), making the same alterations in ChAd68 it was generated the ChAdOx2 (Morris et al., 2016Morris SJ, Sebastian S, Spencer AJ and Gilbert SC (2016) Simian adenoviruses as vaccine vectors. Future Virol 11:649. ).

Even tough neutralization assays are important tools to evaluate inhibition of viral vector transduction efficiency, it was observed that a ChAd68 adenoviral vector modified in the hexon protein resisted neutralization by antisera of animals immunized with WT ChAd68, but failed to transduce target cells and express the transgene, suggesting that neutralization assay may not be a reliable test to predict vector transduction efficiency (Pichla-Gollon et al., 2009Pichla-Gollon SL, Lin S-W, Hensley SE, Lasaro MO, Herkenhoff-Haut L, Drinker M, Tatsis N, Gao G-P, Wilson JM, Ertl HCJ et al. (2009) Effect of preexisting immunity on an adenovirus vaccine vector: In vitro neutralization assays fail to predict inhibition by antiviral antibody in vivo. J Virol 83:5567-5573. ). Induction of antibody response against transgene expression mediated by Ad26 and ChAd6 and ChAd7 were lower in comparison with Ad5, suggesting that Ad5 is more efficient to induce high levels of gene expression and immune response (Chen et al., 2010Chen H, Xiang Z Q, Li Y, Kurupati R K, Jia B, Bian A, Zhou D M, Hutnick N, Yuan S, Gray C, Serwanga J, Auma B, Kaleebu P, Zhou X, Betts M R, Ertl H C J (2010) Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae. J Virol 84: 10522-10532. ). Another interesting data is that use of prime-boost regimens with combination of different adenoviral types did not improve immune response (Weaver et al., 2009Weaver EA, Nehete PN, Buchl SS, Senac JS, Palmer D, Ng P, Sastry KJ and Barry MA (2009) Comparison of replication-competent, first generation, and helper-dependent adenoviral vaccines. PloS One 4:e5059. ). However, monkeys immunized with a combination of Ad26 and Ad5 expressing Gag protein of Simian Immunodeficiency Virus (SIV-Gag) showed increased cellular immune response and survival after SIV challenge (Liu et al., 2009Liu J, O’Brian K, Lynch D, Simmons N, La Porte A, Riggs A, Abbink P, Coffey R, Grandpre L, Seaman M et al. (2009) Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457: 87-91.). Ad5 vectors elicited higher memory T cell activation magnitude, but can also cause functional exhaustion and reduced potency after boost compared to Ad26, Ad35, and Ad48 vectors (Penaloza-MacMaster et al., 2013Penaloza-MacMaster P, Provine NM, Ra J, Borducchi EN, McNally A, Simmons NL, Iampietro MJ and Barouch DH (2013) Alternative serotype adenovirus vaccine vectors elicit memory T cells with enhanced anamnestic capacity compared to Ad5 vectors. J Virol 87:1373-1384. ). This topic will be further discussed in the adenovirus modulation of the immune system session.

Components of the viral particle have also been modified. The introduction of the tripeptide arg-gly-asp (RGD) conferred altered tropism for the viral particle, making it capable of transducing dendritic cells (Worgall et al., 2004Worgall S, Busch A, Rivara M, Bonnyay D, Leopold PL, Merritt R, Hackett NR, Rovelink PW, Bruder JT, Wickham TJ et al. (2004) Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses. J Virol 78:2572-2580. ) and other cells expressing integrins. Replacement of the fiber protein with Sigma 1 from reovirus changed viral tropism to junctional adhesion molecule 1 (JAM1) and sialic acid (Weaver et al., 2012Weaver EA, Mercier GT, Gottschalk S and Barry MA (2012) T-cell-biased immune responses generated by a mucosally targeted adenovirus-σ1 vaccine. Mucosal Immunol 5:311-319. ). The viral particle can also be covered with different compounds to avoid immunologic destruction, using for example alginate microspheres (Sailaja et al., 2002Sailaja G, HogenEsch H, North A, Hays J and Mittal SK (2002) Encapsulation of Recombinant Adenovirus into Alginate Microspheres Circumvents Vector Specific Immune Response. Gene Ther 9:1722. ), or coating with non-immunogenic polymers, such as polyethylene glycol, which reduces vector immunogenicity and protect the virus against neutralizing antibody for persistent gene expression (Prill et al., 2011Prill JM, Espenlaub S, Samen U, Engler T, Schmidt E, Vetrini F, Rosewell A, Grove N, Palmer D, Ng P et al. (2011) Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from kupffer cells. Mol Ther 19:83-92. ; Sun et al., 2019bSun Y, Lv X, Ding P, Wang L, Sun Y, Li S, Zhang H and Gao Z (2019b) Exploring the functions of polymers in adenovirus-mediated gene delivery: Evading immune response and redirecting tropism. Acta Biomater 97:93-104. ). At the same time, adjuvant formulations may increase immunological response in vaccination protocols, and formulations including chitosan and glycol chitosan improve intranasal immunogenicity of Ad5 vector (Gogev et al., 2004Gogev S, de Fays K, Versali MF, Gautier S and Thiry E (2004) Glycol chitosan improves the efficacy of intranasally administrated replication defective human adenovirus type 5 expressing glycoprotein D of bovine herpesvirus 1. Vaccine22:1946-1953. ).

Adenovirus gene therapy for monogenic diseases

The early years

Since the seminal idea of Friedmann and Roblin (1972Friedmann T and Roblin R (1972) Gene therapy for human genetic disease? Science 175: 949-955.) proposing gene therapy to ameliorate human genetic diseases, several experiments have been conducted in situ, in vivo, and ex-vivo to introduce a functional gene or to modulate its expression in a target cell. The use of viral and non-viral vectors for gene delivery and gene editing for permanent correction of patient gene defects are being explored for decades and the promises are starting to become reality (Bulcha et al., 2021Bulcha JT, Wang Y, Ma H, Tai PWL and Gao G (2021) Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther 6:53. ).

Initially, recombinant adenoviral vectors were employed in therapies for common hereditary respiratory diseases, due to their capacity of infecting lung epithelium. The first in vivo therapy used a replication-deficient first-generation adenoviral vector to deliver the alfa-1 antitrypsin gene firstly in lung tissues and then in rat hepatocytes, showing that adenovirus can be used as a vector to treat diseases affecting other sites beyond the lung. The rationale was to convert homozygous mutated hepatocytes cells into heterozygotes, which would not manifest the disease phenotype (Crystal, 1990Crystal RG (1990) α1-Antitrypsin deficiency, emphysema, and liver disease: Genetic basis and strategies for therapy. J Clin Invest 85:1343-1352. ; Rosenfeld et al., 1991Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Pääkkö PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M et al. (1991) Adenovirus-mediated transfer of a recombinant α1-Antitrypsin Gene to the Lung Epithelium in vivo. Science 252:431-434. ; Jaffe et al., 1992Jaffe HA, Danel C, Longenecker G, Metzger M, Setoguchi Y, Rosenfeld MA, Gant TW, Thorgeirsson SS, Stratford-Perricaudet LD, Perricaudet M et al. (1992) Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nat Genet 1:372-378. ). Next, recombinant adenovirus (Ad/CFTR) was employed for gene therapy for cystic fibrosis (CF) through the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. Studies in human bronchial cells (Rich et al., 1993Rich DP, Couture LA, Cardoza LM, Guiggio VM, Armentano D, Espino PC, Hehir K, Welsh MJ, Smith AE and Gregory RJ (1993) Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. Hum Gene Ther 4:461-476. ), human bronchial xenograft model (Engelhardt et al., 1993bEngelhardt JF, Yang Y, Stratford-Perricaudet LD, Allen ED, Kozarsky K, Perricaudet M, Yankaskas JR and Wilson JM (1993b) Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses. Nat Genet 4:27-34. ), and nonhuman primates (Engelhardt et al., 1993aEngelhardt JF, Simon RH, Yang Y, Zepeda M, Weber-Pendleton S, Doranz B, Grossman M and Wilson JM (1993a) Adenovirus-mediated transfer of the C F T R gene to lung of non human primates: biological efficacy study. Hum Gene Ther 769:759-769. ; Goldman et al., 1995Goldman MJ, Litzky LA, Engelhardt JF and Wilson JM (1995) Transfer of the CFTR Gene to the Lung of Nonhuman Primates with E1-Deleted, E2a-Defective Recombinant Adenoviruses: A Preclinical Toxicology Study. Hum Gene Ther6:839-851. ) showed the feasibility and safety of this technology. Even though in the early 1990s there was limited knowledge regarding the safety and effectiveness of gene delivery by first-generation adenovirus vectors in humans, in 1993 it was performed the first clinical trial for human gene therapy with a recombinant adenovirus (AD2/CFTR) in three individulas. The treatment partially corrected the chloride transport defect characteristic of the CF without evidence of adverse effects (Zabner et al., 1993Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE and Welsh MJ (1993) Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 75:207-216. ). In another study, Ad/CFTR was administrated to the nasal and bronchial epithelium of the CF patients. At a high dose, transient systemic inflammation was observed after administration without long-term adverse effects (Crystal et al., 1994Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT and Danel C (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42-51. ). At the same time, other clinical trials were initiated and showed similar results (Zabner et al., 1993Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE and Welsh MJ (1993) Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 75:207-216. , 1996Zabner J, Ramsey BW, Meeker DP, Aitken ML, Balfour RP, Gibson RL, Launspach J, Moscicki RA, Richards SM, Standaert TA et al. (1996) Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin Invest 97:1504-1511. ; Crystal et al., 1994Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT and Danel C (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42-51. ; Zuckerman et al., 1999Zuckerman JB, Robinson CB, McCoy KS, Shell R, Sferra TJ, Chirmule N, Magosin SA, Propert KJ, Brown-Parr EC, Hughes JV et al. (1999) A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis. Hum Gene Ther 10:2973-1985.).

These approaches of gene therapy for genetic diseases seemed promising until 1999 when a patient died after treatment with a second-generation Ad5 vector carrying the human ornithine transcarboxylase (OTC) cDNA for OTC deficiency (Raper et al., 2003Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, Wilson JM and Batshaw ML (2003) Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80:148-158. ). The 18 years old patient was the only one among other 17 OTC deficient patients who died 96 h after gene transfer due to a systemic inflammatory response syndrome. The other patients experienced only flu-like symptoms. A recent study showed that the presence of a complex of pre-existing Ad5 antibodies and the Ad-therapeutic vector could enhance vector transduction and activation of dendritic cells, which may have contributed to the systemic lethal inflammation of that patient (Somanathan et al., 2020Somanathan S, Calcedo R and Wilson JM (2020) Adenovirus-Antibody Complexes Contributed to Lethal Systemic Inflammation in a Gene Therapy Trial. Mol Ther 28:784-793. ). This adverse result rocked the gene therapy research and delayed advances for some time.

The use of HC-Ad was able to overcome some of the limitations of first and second-generation vectors and was employed in some strategies. In nonhuman primate models, the expression of the baboon alpha-fetoprotein transgene delivered by a HC-Ad persisted up to 7 years without adverse effects, declining to about 10%/year (Brunetti-Pierri et al., 2013Brunetti-Pierri N, Ng T, Iannitti D, Cioffi W, Stapleton G, Law M, Breinholt J, Palmer D, Grove N, Rice K et al. (2013) Transgene expression up to 7 years in nonhuman primates following hepatic transduction with helper-dependent adenoviral vectors. Hum Gene Ther 4:761-765. ). In a mouse model of primary kidney disease hyperoxaluria type 1, HC-Ad transferred the alanine-glyoxylate aminotransferase gene under control of a liver-specific promoter, improving the clinical condition of the animals for at least 24 weeks (Castello et al., 2016Castello R, Borzone R, D’Aria S, Annunziata P, Piccolo P and Brunetti-Pierri N (2016) Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1. Gene Ther 23:129-134. ). In another study, primary dystrophin-deficient mouse myoblasts were successfully transduced with an adenoviral vector carrying the full-length murine dystrophin cDNA under control of a muscle-specific promoter and a lacZ reporter construct (Kochanek et al., 1996Kochanek S, Clemens PR, Mitani K, Chen HH, Chan S and Caskey CT (1996) A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and β-galactosidase. Proc Natl Acad Sci U S A 93:5731-5736. ).

Pre-clinical and clinical trials

Next, we present pre-clinical and clinical results of some monogenic disease therapies using adenoviral vectors. Hemophilia A and B gene therapy has been investigated since the 1990’s (High, 2003High KA(2003) Gene transfer as an approach to treating hemophilia. Semin Thromb Hemost29:107-120. ), they are X-linked genetic diseases caused by mutations in the coagulation factors XIII and IX genes, respectively. The portal infusion of a first-generation Ad with the canine factor IX gene transiently corrected (1-2 months) the canine hemophilia B (Kay et al., 1994Kay MA, Landen CN, Rothenberg SR, Taylor LA, Leland F, Wiehle S, Fang B, Bellinger D, Finegold M, Thompson AR et al. (1994) In vivo hepatic gene therapy: Complete albeit transient correction of factor IX deficiency in hemophilia B dogs. Proc Natl Acad Sci U S A 91:2353-2357. ). However, longer expression (5 months) of the beta domain of factor VIII was observed after lower doses of Ad administration to correct mice hemophilia A (Connelly et al., 1996Connelly S, Gardner JM, Lyons RM, McClelland A and Kaleko M (1996) Sustained expression of therapeutic levels of human factor VIII in mice. Blood 87:4671-4677. ). In following studies using a HC-Ad, the correction of canine hemophilia B and A without toxicity or thrombocytopenia was obtained (Chuah et al., 2003Chuah MKL, Schiedner G, Thorrez L, Brown B, Johnston M, Gillijns V, Hertel S, Van Rooijen N, Lillicrap D, Collen D et al. (2003) Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia a following gene therapy with high-capacity adenoviral vectors. Blood 101:1734-43. ; Ehrhardt et al., 2003Ehrhardt A, Xu H, Dillow AM, Bellinger DA, Nichols TC and Kay MA (2003) A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia. Blood 102:2403-2411. ). Interestingly, mice neonatal gene therapy to express factor VIII lasted for one year, even with the quick decline of its levels. Despite re-administration of the HC-Ad was well tolerated, immunity to adenovirus persisted (Hu et al., 2011Hu C, Cela RG, Suzuki M, Lee B and Lipshutz GS (2011) Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII. Proc Natl Acad Sci U S A 108:2082-2087. ). An option for long-term expression of these genes was the use of a transposase for the therapeutic gene integration. For hemophilia B, a HC-Ad stabilized through the Sleepy beauty transposase (SB) showed sustained expression of human coagulation factor IX for more than six months in mice (Yant et al., 2002Yant SR, Ehrhardt A, Mikkelsen JG, Meuse L, Pham T and Kay MA (2002) Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat Biotechnol 20:999-1005. ). A hyperactive SB (SB100X) corrected hemophilia B in mice and canine models by somatic integration in the liver (Hausl et al., 2010Hausl MA, Zhang W, Müther N, Rauschhuber C, Franck HG, Merricks EP, Nichols TC, Kay MA and Ehrhardt A (2010) Hyperactive sleeping beauty transposase enables persistent phenotypic correction in mice and a canine model for hemophilia B. Mol Ther 18:1896-1906. ).

Only one clinical study used a HC-Ad expressing the B domain of factor VIII under albumin promoter for liver-specific expression. The study was stopped because the first patient developed systemic side effects, probably due to the high production of inflammatory cytokines and the factor VIII levels were about 1% (Mannucci, 2002Mannucci PM (2002) Hemophilia and related bleeding disorders: a story of dismay and success. Hematology Am Soc Hematol Educ Program 2002:1-9. ).

Regarding CF gene therapy, in a CFTR-knockout mouse model, the in-uterus expression of Cftr mediated by an Ad vector did not improve the survival of the animals (Davies et al., 2008Davies LA, Varathalingam A, Painter H, Lawton AE, Sumner-Jones SG, Nunez-Alonso GA, Chan M, Munkonge F, Alton EW, Hyde SC et al. (2008) Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice. Mol Ther 16:812-818. ). One of the problems is that CAR localizes to the basal membrane of the airway epithelium, thus limiting the capacity of the Ad vector to transduce the target cells (Walters et al., 1999Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ and Zabner J (1999) Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem 274:10219-10226.). The use of lysophosphatidylcholine (LPC) formulation during the application of HC-Ad to the lung facilitated access to CAR and improved the gene transfer efficiency of mice, pigs, and ferrets’ epithelia. (Yan et al., 2015Yan Z, Stewart ZA, Sinn PL, Olsen JC, Hu J, McCray PB and Engelhardt JF (2015) Ferret and pig models of cystic fibrosis: prospects and promise for gene therapy. Hum Gene Ther Clin Dev 26:38-49.; Cao et al., 2018Cao H, Ouyang H, Grasemann H, Bartlett C, Du K, Duan R, Shi F, Estrada M, Seigel KE, Coates AL et al. (2018) Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy. Hum Gene Ther 29:643-652.). In another study, the treatment with the pharmacological drug cyclophosphamide was shown to overcome the immunological response to HC-Ad-CFTR allowing the sustained expression of Cftr when the vector was repeatedly delivered to the mouse airways. The treatment reduced the expression of T cells and their infiltration into mouse lung tissues, as well as adenovirus antibody and neutralizing activity (Cao et al., 2020Cao H, Duan R and Hu J (2020) Overcoming immunological challenges to helper-dependent adenoviral vector-mediated long-term CFTR expression in mouse airways. Genes 11:565.). The incorporation of the transposons piggyBac into the HC-Ad led to efficient expression of the transgene in pig´s lungs (Cooney et al., 2018Cooney AL, Singh BK, Loza LM, Thornell IM, Hippee CE, Powers LS, Ostedgaard LS, Meyerholz DK, Wohlford-Lenane C, Stoltz DA et al. (2018) Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus. Nucleic Acids Res 46:9591-9600. ). In 2015 the largest clinical trial liposomes-mediated delivery of the CFTR gene showed a modest stabilization of the lung function but not sufficient to improve lung function. Consequently, development of efficient vectors that are able to transduce lung cells and animal models for CF gene therapy are still needed (Alton et al., 2015Alton EWFW, Armstrong DK, Ashby D, Bayfield KJ, Bilton D, Bloomfield E., Boyd AC, Brand J, Buchan R, Calcedo R et al. (2015) Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: A randomised, double-blind, placebo-controlled, phase 2b trial. Lancet Respir Med 3:684-691. ; Yan Z et al., 2019Yan Y, Xu H, Wang J, Wu X, Wen W, Liang Y, Wang L, Liu F and Du X (2019) Inhibition of breast cancer cells by targeting E2F-1 gene and expressing IL15 oncolytic adenovirus. Biosci Rep 39:BSR20190384. ).

Gene therapy using adenovirus has been attractive for the treatment of liver diseases because of the many metabolic functions of the liver, the hepatocyte Ad tropism, and the high capacity to produce and secrete proteins in circulation (Maestro et al., 2021Maestro S, Weber ND, Zabaleta N, Aldabe R and Gonzalez-Aseguinolaza G (2021) Novel vectors and approaches for gene therapy in liver diseases. JHEP Reports 3:100300. ). In a model of neonatal bovine citrullinemia, an inborn error of metabolism caused by the deficiency of argininosuccinate synthetase (ASS) that leads to hyperammonemia, the systematic administration of a first-generation Ad human ASS allowed the liver transduction and partially corrected the defect (Lee et al., 1999Lee B, Dennis JA, Healy PJ, Mull B, Pastore L, Yu H, Aguilar-Cordova E, O’Brien W, Reeds P and AL Beaudet et al. (1999) Hepatocyte gene therapy in a large animal: A neonatal bovine model of citrullinemia. Proc Natl Acad Sci U S A 96:3981-3986. ). The deficiency of ornithine transcarbamylase (OTC) is another liver disease, X-linked, associated with the urea cycle that leads to hyperammonemia encephalopathy. A mouse model with an earlier Ad vector and CMV promoter corrected the Otc deficiency for two months (Ye et al., 1996Ye X, Robinson MB, Batshaw ML, Furth EE, Smith I and Wilson JM (1996) Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. J Biol Chem 271:3639-3646. ). Combining HC-Ad, specific tissue promoter, and post-transcriptional enhancement sequences allowed overexpression of Otc and long-term correction of the deficiency in mice without toxicity (Mian et al., 2004Mian A, McCormack WM, Mane V, Kleppe S, Ng P, Finegold M, O’Brien WE, Rodgers JR, Beaudet AL and Lee B (2004) Long-term correction of ornithine transcarbamylase deficiency by WPRE-mediated overexpression using a helper-dependent adenovirus. Mol Ther 10:492-499. ). However, after the first clinical trials for OTC and the fatal outcome described above (Raper et al., 2003Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, Wilson JM and Batshaw ML (2003) Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80:148-158. ), no other clinical trials for OTC with adenovirus have been conducted.

Adenoviral vectors, more specially HC-Ads, are widely used as experimental therapeutic vectors, but in clinical trials for genetic diseases, most promisor gene therapy is by using adeno-associated virus or lentiviral vectors. Several phases 1, 2 and 3 clinical trials for replacement therapy have been concluded or are ongoing for hemophilia (Perrin et al., 2019Perrin GQ, Herzog RW and Markusic DM (2019) Update on clinical gene therapy for hemophilia. Blood 133:407-414. ; Batty and Lillicrap, 2021Batty P and Lillicrap D (2021) Hemophilia gene therapy: Approaching the first licensed product. Hemasphere 5:e540. ), CF (Guggino and Cebotaru, 2020Guggino WB and Cebotaru L (2020) Gene Therapy for Cystic Fibrosis Paved the Way for the Use of Adeno-Associated Virus in Gene Therapy. Hum Gene Ther 31:538-541. ), Pompe (Unnisa et al., 2022Unnisa Z, Yoon JK, Schindler JW, Mason C and van Til NP (2022) Gene Therapy Developments for Pompe Disease. Biomedicines 10:302. ) and other diseases (see ClinicalTrials.govClinicalTrials.gov, gov, https://clinicaltrials.gov/ (accessed 21 June 2022).
https://clinicaltrials.gov/... ).

Ex-vivo and gene editing

The ex-vivo gene therapy consists of modified cells outside the body to express a therapeutic gene and subsequently implant them back into patients. This therapy has been useful for inherited rare blood disorders e.g beta-thalassemia, sickle cell disease (SCD), and other hematological diseases. In these cases, the patient’s hematopoietic stem cells (HSC) are collected, transduced with a vector carrying the therapeutic gene and injected back into the patients (Tambuyzer et al., 2020Tambuyzer E, Vandendriessche B, Austin CP, Brooks PJ, Larsson K, Miller Needleman KI, Valentine J, Davies K, Groft SC, Preti R et al. (2020) Therapies for rare diseases: therapeutic modalities, progress and challenges ahead. Nat Rev Drug Discov 19:93-111. ). This technique has several challenges including insufficient HSC obtained from the patient, genotoxicity and limitations of the viral vectors, loss of HSC multipotency during ex vivo manipulation, reduced number of transduced cells for reimplantation, technical complexity and high cost (Li and Lieber, 2019Li C and Lieber A (2019) Adenovirus vectors in hematopoietic stem cell genome editing. FEBS Letters 593:3623-3648. ; Telen et al., 2019Telen MJ, Malik P and Vercellotti GM (2019) Therapeutic strategies for sickle cell disease: towards a multi-agent approach. Nat Rev Drug Discov 18:139-158. ). However, in the last years great advances in this area have been made.

The ex-vivo Ad transduction into human conjunctival epithelium and cornea, showed sustained expression of reporter genes, interleukin 10 and others, suggesting that this strategy could be employed to suppress immune-mediated disorders (Oral et al., 1997Oral HB, Larkin DFP, Fehervari Z, Byrnes AP, Rankin AM, Haskard DO, Wood MJA, Dallman MJ and George AJT (1997) Ex vivo adenovirus-mediated gene transfer and immunomodulatory protein production in human cornea. Gene Ther 4:639-647. ; Shen et al., 2001Shen J, Taylor N, Duncan L, Kovesdi I, Bruder JT, Forrester JV and Dick AD (2001) Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium. Br J Ophthalmol 85:861-867. ; Qian et al., 2004Qian Y, Leong FL, Kazlauskas A and Dana MR (2004) Ex vivo adenovirus-mediated gene transfer to corneal graft endothelial cells in mice. Invest Ophthalmol Vis Sci 45:2187-2193. ). For Sickle cell disease (SCD), a monogenic disorder caused by a mutation in the beta-globin gene (beta S allele) compromising the production of normal adult hemoglobin (Vichinsky et al., 2000Vichinsky EP, Neumayr LD, Earles AN, Williams R, Lennette ET, Dean D, Nickerson B, Orringer E, McKie V, Bellevue R et al. (2000) Causes and outcomes of the acute chest syndrome in sickle cell disease. National Acute Chest Syndrome Study Group. N Engl J Med 342:1855-1865.), gene therapy approaches include the ex-vivo transduction of the HSPC for expressing the intact beta-globin gene, anti-sickling beta-globin, or the fetal gamma-globin. In mice models, ex-vivo HSPC transduction of a HC-Ad5/35 vector carrying SB100x transposase-mediated gamma-globin gene and transplantation into irradiated mice reached 95% of gamma-globin-positive peripheral red blood cells (Wang et al., 2020Wang H, Georgakopoulou A, Li C, Liu Z, Gil S, Bashyam A, Yannaki E, Anagnostopoulos A, Pande A, Izsvák Z et al. (2020) Curative in vivo hematopoietic stem cell gene therapy of murine thalassemia using large regulatory elements. JCI Insight 5:e139538. ). This result complemented the in vivo model that resulted in an incomplete correction of the thalassemia phenotype in mice (Wang H et al., 2019Wang H, Georgakopoulou A, Psatha N, Li C, Capsali C, Samal HB, Anagnostopoulos A, Ehrhardt A, Izsvák Z, Papayannopoulou T et al. (2019) In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia. J Clin Invest 129:598-615. ; Wang et al., 2020Wang H, Georgakopoulou A, Li C, Liu Z, Gil S, Bashyam A, Yannaki E, Anagnostopoulos A, Pande A, Izsvák Z et al. (2020) Curative in vivo hematopoietic stem cell gene therapy of murine thalassemia using large regulatory elements. JCI Insight 5:e139538. ).

Gene-addition strategies have been optimized over the past few decades and the genome editing tools based on clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) are being widely used for modifying HSC and other cells genome for gene therapy (Maggio et al., 2016Maggio I, Liu J, Janssen JM, Chen X and Gonçalves MAFV (2016) Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells. Sci Rep 6:37051. ; Yin et al., 2017Yin H, Kauffman KJ and Anderson DG (2017) Delivery technologies for genome editing. Nat Rev Drug Discov 16:387-399. ; Stephens et al., 2019Stephens CJ, Lauron EJ, Kashentseva E, Lu ZH, Yokoyama WM and Curiel DT (2019) Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. J Control Release 298:128-141. ; Bandara et al., 2021Bandara RA, Chen ZR and Hu J (2021) Potential of helper-dependent Adenoviral vectors in CRISPR-cas9-mediated lung gene therapy. Cell Biosci 11:145. ). These strategies precisely target the gene of interest and can fix or “cure” the disease. However, a hurdle to be overcome is the off-target activities on unintended sites. A study showed a low scarless homology-directed genome editing of the modified cells by applying these nucleases together with an Ad donor DNA delivery compared with lentiviral or non-viral vectors templates (Holkers et al., 2014Holkers M, Maggio I, Henriques SFD, Janssen JM, Cathomen T and Gonçalves MAFV (2014) Adenoviral vector DNA for accurate genome editing with engineered nucleases. Nat Methods 11:1051-1057. ).

The reactivation of the fetal hemoglobin HbF coded by the gamma-globin gene by knocking out its repressor BCL-11A (Brendel et al., 2016Brendel C, Guda S, Renella R, Bauer DE, Canver MC, Kim YJ, Heeney MM, Klatt D, Fogel J, Milsom MD et al. (2016) Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype. J Clin Invest 126:3868-3878. ; Li et al., 2018aLi C, Psatha N, Sova P, Gil S, Wang H, Kim J, Kulkarni C, Valensisi C, Hawkins RD, Stamatoyannopoulos G, Lieber A et al. (2018a) Reactivation of g-globin in adult b-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing. Blood 131:2915-2928. ) or the binding sites in the globin gene (Traxler et al., 2016Traxler EA, Yao Y, Wang YD, Woodard KJ, Kurita R, Nakamura Y, Hughes JR, Hardison RC, Blobel GA, Li C et al. (2016) A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 22:987-990. ), and the correction of the beta S mutation has been the strategy for the CRISPR/Cas9-mediated gene therapy for SCD (Dever et al., 2016Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S et al. (2016) CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539:384-389. ). Recently, a HC-Ad5/35 vector expressing the CRISPR/Cas9 platform (Li et al., 2018bLi C, Psatha N, Wang H, Singh M, Samal HB, Zhang W, Ehrhardt A, Izsvák Z, Papayannopoulou T and Lieber A (2018b) Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies. Mol Ther Methods Clin Dev 9:142-152. ) repressed the binding region within the gamma-globin promoter after transduction of HSPCs from a thalassemic mice models (Li et al., 2021Li C, Wang H, Georgakopoulou A, Gil S, Yannaki E and Lieber A (2021) In vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model. Mol Ther 29:822-837. ). The transplantation of the modified HSCs into the irradiated animal as well as the in vivo intravenous injection of the vector into the mice showed efficient target site disruption and relevant switch from human beta- to gamma-globin expression that was sustained after a secondary transplantation of HSPCs, without observed hematological abnormalities in the long-term follow up (Li et al., 2021Li C, Wang H, Georgakopoulou A, Gil S, Yannaki E and Lieber A (2021) In vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model. Mol Ther 29:822-837. ).

In an ex-vivo approach for hemophilia B, a HC-Ad5 vector containing an inducible gene-specific CRISPR/Cas9 system together with an adeno-associated virus containing the modified donor, and a HC-Ad5 vector with all the components were used to transduce liver cell lines stably expressing mutated canine factor IX gene (carrying a point mutation). Interestingly, the single vector showed 6% of efficiency, which was superior to the two-vector strategy, thus CRISPR/CAS9 viral vector delivery is promising for the correction of mutated factor IX in disease models (Gao et al., 2019Gao J, Bergmann T, Zhang W, Schiwon M, Ehrke-Schulz E and Ehrhardt A (2019) Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an in vitro Model for Canine Hemophilia B. Mol Ther Nucleic Acids 14:364-376. ).

Despite some hurdles, advances in in-vivo, ex-vivo, and genome edition using adenovirus-delivery as a single vector (Palmer et al., 2020Palmer DJ, Turner DL and Ng P (2020) A Single “All-in-One” Helper-Dependent Adenovirus to Deliver Donor DNA and CRISPR/Cas9 for Efficient Homology-Directed Repair. Mol Ther Methods Clin Dev 17:441-447. ) or combined (Lino et al., 2018Lino CA, Harper JC, Carney JP and Timlin JA (2018) Delivering CRISPR: A review of the challenges and approaches. Drug Deliv 25:1234-1257. ) with other vectors are promising for genetic diseases gene therapy and may provide more gene therapy products in the near future.

Adenovirus in cancer gene therapy

Cancer is a disease characterized by genetic alterations, uncontrolled cell functions, and loss of original cell characteristics (Hanahan and Weinberg, 2000Hanahan D and Weinberg RA (2000) The Hallmarks of Cancer. Cell100:57-70.). In 2020, it was estimated 19.3 million new cases and 10 million cancer deaths worldwide (Sung et al., 2021Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209-249. ), it is considered one of the leading causes of death in the world (Bray et al., 2021Bray F, Laversanne M, Weiderpass E and Soerjomataram I (2021) The ever-increasing importance of cancer as a leading cause of premature death worldwide. Cancer 127:3029-3030. ). Despite all acquired knowledge, cancer treatment is still a challenge for several types of tumors (Wang et al., 2018Wang JJ, Lei KF and Han F (2018) Tumor microenvironment: Recent advances in various cancer treatments. Eur Rev Med Pharmacol Sci 22:3855-3864. ). Conventional therapy based on chemo- and radiotherapy alone are not always successful (Wang et al., 2018Wang JJ, Lei KF and Han F (2018) Tumor microenvironment: Recent advances in various cancer treatments. Eur Rev Med Pharmacol Sci 22:3855-3864. ). Hanahan and Weinberg have described the hallmarks of cancer and each one of them is a relevant factor in tumor development and are key targets for cancer therapy (Hanahan and Weinberg, 2000Hanahan D and Weinberg RA (2000) The Hallmarks of Cancer. Cell100:57-70., 2011Hanahan D and Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell 144:646-674. ; Hanahan, 2022Hanahan D (2022) Hallmarks of Cancer: New Dimensions. Cancer Discov 12:31-46. ). Gene therapy using adenoviral vectors appears to be an interesting option for treating cancer as it can restore or inhibit pathways that were lost or modified during tumorigenesis (Sun et al., 2019aSun W, Shi Q, Zhang H, Yang K, Ke Y, Wang Y and Qiao L (2019a) Advances in the techniques and methodologies of cancer gene therapy. Discov Med 27:45-55.). Most of the approaches described in this section employed first generation adenoviral vectors or oncolytic adenoviruses. In Figure 2 we show a summary view of different approaches of gene therapy in cancer treatment.

Targeting cell proliferation and growth suppressors evasion

One of the most remarkable tumor cell characteristics is the ability of uncontrolled proliferation (Hanahan and Weinberg, 2011Hanahan D and Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell 144:646-674. ). Modulation of pathways and genes that control processes involved in cell cycle, proliferation, growth, and survival are commonly seen in cancer, mainly related to tumor suppressors’ inhibition and oncogenes activation (Park et al., 2020Park JH, Pyun WY and Park HW (2020) Cancer Metabolism: Phenotype, Signaling and Therapeutic Targets. Cells 9:2308. ).

The search for reestablishing phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway normal regulation is one of the adenovirus gene therapy aims. This pathway in normal cells induces cell growth and proliferation and inhibits apoptosis (Fresno Vara et al., 2004Fresno Vara JÁ, Casado E, de Castro J, Cejas P, Belda-Iniesta C and González-Barón M (2004) P13K/Akt signalling pathway and cancer. Cancer Treat Rev 30:193-204. ). Different alterations contribute to PI3K/AKT pathway constitutive activation in cancer, including rat sarcoma virus proto-oncogene (RAS) constitutive activation, and loss of the pathway negative regulator, phosphatase and tensin homolog (PTEN) (Fresno Vara et al., 2004Fresno Vara JÁ, Casado E, de Castro J, Cejas P, Belda-Iniesta C and González-Barón M (2004) P13K/Akt signalling pathway and cancer. Cancer Treat Rev 30:193-204. ; Hanahan and Weinberg, 2011Hanahan D and Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell 144:646-674. ; Santarpia et al., 2012Santarpia L, Lippman SM and El-Naggar AK (2012) Targeting the MAPKRASRAF signaling pathway in cancer therapy. Expert Opin Ther Targets 16:103-119. ; Park et al., 2020Park JH, Pyun WY and Park HW (2020) Cancer Metabolism: Phenotype, Signaling and Therapeutic Targets. Cells 9:2308. ).

Induction of PTEN expression mediated by adenovirus (Ad-PTEN) in several types of cancer demonstrated to be effective to downregulate PI3K/AKT pathway, consequently contributing to apoptosis induction, migration, and growth inhibition in tumor cell lines and tumor suppression in vivo (see summary data in ^{Table S1} Table S1 - Adenoviral vectors modulating proliferation and survival of tumor cells. ). Nonetheless, this effect is more effective in cell lines with loss or mutated PTEN in comparison to tumor cells carrying WT PTEN (Tanaka and Grossman, 2003Tanaka M and Grossman HB (2003) In vivo gene therapy of human bladder cancer with PTEN suppresses tumor growth, downregulates phosphorylated Akt, and increases sensitivity to doxorubicin. Gene Ther 10:1636-1642. ; Hamada et al., 1999Hamada H, Yokoyama T, Furukawa T, Sato S, Yajima A, Sato M, Fujimura S and Horii A (1999) Adenovirus-mediated delivery of the PTEN gene inhibits cell growth by induction of apoptosis in endometrial cancer. Int J Oncol 15:1069-1074; Tanaka et al., 2005; Rosser et al., 2004Rosser CJ, Tanaka M, Pisters LL, Tanaka N, Levy LB, Hoover DC, Barton Grossman H, McDonnell TJ, Kuban DA and Meyn RE (2004) Adenoviral-mediated PTEN transgene expression sensitizes Bcl-2-expressing prostate cancer cells to radiation. Cancer Gene Ther 11:273-279. ). Different studies analyzed the combination of Ad-PTEN with other therapeutic agents to potentialize its antitumor effect. Ad-PTEN enhanced the doxorubicin efficacy in bladder and prostate cancer (Tanaka and Grossman, 2003Tanaka M and Grossman HB (2003) In vivo gene therapy of human bladder cancer with PTEN suppresses tumor growth, downregulates phosphorylated Akt, and increases sensitivity to doxorubicin. Gene Ther 10:1636-1642. ; Tanaka et al., 2005) and sensitized tumor cells to cisplatin (Li D et al., 2013Li Y, Zhang B, Zhang H, Zhu X, Feng D, Zhang D, Zhuo B, Li L and Zheng J (2013) Oncolytic adenovirus armed with shRNA targeting MYCN gene inhibits neuroblastoma cell proliferation and in vivo xenograft tumor growth. J Cancer Res Clin Oncol 139:933-941. ; Wu et al., 2015Wu S, Zhang Z, Yu R, Zhang J, Liu Y, Song X, Yi S, Liu J, Chen J, Yin Y et al. (2014) Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity. Clin Vaccine Immunol 21:156-164. ), docetaxel (Liu Z et al., 2012Liu J, Fang L, Cheng Q, Li L, Su C, Zhang B, Pei D, Yang J, Li W and Zheng J (2012) Effects of G250 promoter controlled conditionally replicative adenovirus expressing Ki67-siRNA on renal cancer cell. Cancer Sci 103:1880-1888. ), to a PI3K inhibitor (Ren et al., 2012Ren Y, Zhou X, Qi Y, Li G, Mei M and Yao Z (2012) PTEN activation sensitizes breast cancer to PI3-kinase inhibitor through the β-catenin signaling pathway. Oncol Rep 28:943-948. ), radiotherapy (Pappas et al., 2007Pappas G, Zumstein LA, Munshi A, Hobbs M and Meyn RE (2007) Adenoviral-mediated PTEN expression radiosensitizes non-small cell lung cancer cells by suppressing DNA repair capacity. Cancer Gene Ther 14:543-549; Rosser et al., 2004), to TIMP-2 (Lu et al., 2004Lu W, Zhou X, Hong B, Liu J and Yue Z (2004) Suppression of invasion in human U87 glioma cells by adenovirus-mediated co-transfer of TIMP-2 and PTEN gene. Cancer Lett 214:205-213.) and caffeine (Saito et al., 2003Saito Y, Gopalan B, Mhashilkar AM, Roth JA, Chada S, Zumstein L and Ramesh R (2003) Adenovirus-mediated PTEN treatment combined with caffeine produces a synergistic therapeutic effect in colorectal cancer cells. Cancer Gene Ther 10:803-813. ). To provide specificity to tumor cells, PTEN has been conjugated to the epithelial cell adhesion molecule (EpCAM), which resulted in better antitumor effects in liver cancer in vivo and in vitro (Liu Z et al., 2018Liu FR, Bai S, Feng Q, Pan XY, Song SL, Fang H, Cui J and Yang JL (2018) Anti-colorectal cancer effects of anti-p21Ras scFv delivered by the recombinant adenovirus KGHV500 and cytokine-induced killer cells. BMC Cancer 18:1087. ). Oncolytic adenoviruses expressing PTEN under control of a specific promoter to prostate cancer have conferred almost complete tumor regression and high specificity in prostate cancer in vitro and in a murine model (Ding et al., 2012Ding M, Cao X, Xu HN, Fan JK, Huang HL, Yang DQ, Li YH, Wang J, Li R, Liu XY (2012) Prostate cancer-specific and potent antitumor effect of a DD3-controlled oncolytic virus harboring the PTEN gene. PloS One 7:e35153. ).

Meanwhile, different studies have focused on adenoviral gene therapy for RAS blockage. The RAS gene family (H-RAS, K-RAS, and N-RAS) is one of the most altered genes in cancer (Zinatizadeh et al., 2019Zinatizadeh MR, Momeni SA, Zarandi PK, Chalbatani GM, Dana H, Mirzaei HR, Akbari ME and Miri SR (2019) The role and function of ras-association domain family in cancer: A review. Genes Dis 6:378-384.), which is involved in proliferation, survival, angiogenesis, and cell motility (Santarpia et al., 2012Santarpia L, Lippman SM and El-Naggar AK (2012) Targeting the MAPKRASRAF signaling pathway in cancer therapy. Expert Opin Ther Targets 16:103-119. ). Several strategies using adenovirus have been employed in an attempt to decrease RAS activity in cancer, such as the expression of neutralizing anti-RAS antibody (van Etten et al., 2002van Etten B, ten Hagen T, de Vries M, Ambagtsheer G, Huet T and Eggermont A (2002) Prerequisites for effective adenovirus mediated gene therapy of colorectal liver metastases in the rat using an intracellular neutralizing antibody fragment to p21-Ras. Br J Cancer 86:436-442. ; Yang et al., 2016Yang JL, Pan XY, Zhao WX, Hu QC, Ding F, Feng Q, Li GY and Luo Y (2016) The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo. Int J Oncol 48:1218-1228. ), gene silencing using antisense and small interference RNAs (siRNA) (Nakano et al., 2001Nakano M, Aoki K, Matsumoto N, Ohnami S, Hatanaka K, Hibi T, Terada M and Yoshida T (2001) Suppression of colorectal cancer growth using an adenovirus vector expressing an antisense K-ras RNA. Mol Ther 3:491-499. ; Chen et al., 2005Chen L-M, Le H-Y, Qin R-Y, Kumar M, Du Z-Y, Xia R-J and Deng J (2005) Reversal of the phenotype by K-ras val12 silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1. World J Gastroenterol 11:831-838.; Zhang et al., 2006Zhang Z, Jiang G, Yang F and Wang J (2006) Knockdown of mutant K-ras expression by adenovirus-mediated siRNA inhibits the in vitro and in vivo growth of lung cancer cells. Cancer Biol Ther 5:1481-1486. ), induction of a dominant-negative mutant form of RAS (Senmaru et al., 1998Senmaru N, Shichinohe T, Takeuchi M, Miyamoto M, Sazawa A, Ogiso Y, Takahashi T, Okushiba S, Takimoto M, Kato H et al. (1998) Suppression of Erk Activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y. Int J Cancer 78:366-371; Watanabe et al., 2001Watanabe T, Shinohara N, Sazawa A, Takimoto M, Hashimoto A, Koyanagi T and Kuzumaki N (2001) Adenovirus-mediated gene therapy for bladder cancer in an orthotopic model usind a dominant negative H-Ras mutant. Int J Cancer 92:712-717.; Stoll et al., 2005Stoll V, Calleja V, Vassaux G, Downward J and Lemoine NR (2005) Dominant negative inhibitors of signalling through the phosphoinositol 3-kinase pathway for gene therapy of pancreatic cancer. Gut 54:109-116. ), and ribozymes against RAS (Irie et al., 1999Irie A, Anderegg B, Kashani-Sabet M, Ohkawa T, Suzuki T, Halks-Miller M, Curiel DT and Scanlon1 KJ (1999) Therapeutic Efficacy of an Adenovirus-Mediated Anti-H-ras Ribozyme in Experimental Bladder Cancer. Antisense Nucleic Acid Drug Dev 9:341-349.; Tsuchida et al., 2000Tsuchida T, Kijima H, Hori S, Oshika Y, Tokunaga T, Kawai K, Yamazaki H, Ueyama Y, Scanlon KJ, Tamaoki N et al. (2000) Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma. Cancer Gene Ther 7:373-383.; Zhang et al., 2000Zhang Y-A, Nemunaitis J, Scanlon KJ and Tong AW (2000) Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice. Gene Ther 7:2041-2050.; Wang et al., 2002Wang C-H, Tsai L-J, Tsao Y-P, Hsieh J-T, Chien W-W, Liao C-L, Wang H-W, Liu H-S and Chen S-L (2002) Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase-and mitochondria-dependent pathways. Bioochem Biophys Res Commun 298:805-814.) resulting in antitumor effects in vivo and in vitro (^{Table S1} Table S1 - Adenoviral vectors modulating proliferation and survival of tumor cells. ). Combinatory treatment using cytokines and RAS-targeted therapy resulted in synergistic tumor inhibition, as seen in pancreatic cancer using Interferon-alpha (IFN-α) (Hatanaka et al., 2004Hatanaka K, Suzuki K, Miura Y, Yoshida K, Ohnami S, Kitade Y, Yoshida T and Aoki K (2004) Interferon-α and antisense K-ras RNA combination gene therapy against pancreatic cancer. J Gene Med6:1139-1148. ) and in colon cancer, with Interleukin-27 (IL-27) (Lebedeva et al., 2007Lebedeva IV, Su ZZ, Emdad L, Kolomeyer A, Sarkar D, Kitada S, Waxman S, Reed JC and Fisher PB (2007) Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells. Oncogene 26:733-744. ). Additionally, oncolytic adenovirus expressing anti-RAS antibody conferred specificity and high antitumor efficacy in cell lines from different cancer types (Pan et al., 2017Pan XY, Liu XJ, Li J, Zhen SJ, Liu DX, Feng Q, Zhao WX, Luo Y, Zhang YL, Li HW et al. (2017) The antitumor efficacy of anti-p21Ras scFv mediated by the dual-promoter-regulated recombinant adenovirus KGHV300. Gene Ther 24:40-48.). Another approach used for targeting tumor cells was the use of cytokine-induced killer (CIK) cells as vehicles for adenoviral delivery. CIK cells carrying adenovirus expressing anti-RAS antibody guaranteed tumor specificity in glioma, lung, and colon cancer (Liu et al., 2018Liu Z, Sun X, Xiao S, Lin Y, Li C, Hao N, Zhou M, Deng R, Ke S and Zhong Z (2018) Characterization of aptamer-mediated gene delivery system for liver cancer therapy. Oncotarget 9: 6830-6840.; Lin et al., 2019Lin XR, Zhou XL, Feng Q, Pan XY, Song SL, Fang H, Lei J and Yang JL (2019) CIK cell-based delivery of recombinant adenovirus KGHV500 carrying the anti-p21Ras scFv gene enhances the anti-tumor effect and safety in lung cancer. J Cancer Res Clin Oncol 145:1123-1132. ; Qian et al., 2021Qian J, Yang M, Feng Q, Pan XY, Yang LL and Yang JL (2021) Inhibition of glioma by adenovirus KGHV500 encoding anti-p21Ras scFv and carried by cytokine-induced killer cells. Exp Biol Med 246:1228-1238. ). Although, in a liver cancer model, CIK cells delivery did not demonstrate tumor specificity and adenoviruses were detected in different organs, even though antitumor activity was achieved (Dai et al., 2021Dai F, Zhang PB, Feng Q, Pan XY, Song SL, Cui J and Yang JL (2021) Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer. J Cancer 12:2768-2776. ).

The retinoblastoma pathway (pRb) has also been a target of adenovirus cancer gene therapy. pRb inhibits proliferation by direct interaction with the transcription factor, E2 promoter binding factor (E2F). It releases E2F to trigger the cell cycling when it is phosphorylated and P16, known to be a tumor suppressor gene, prevents pRb phosphorylation and therefore cell cycle progression (D’Arcangelo et al., 2017D’Arcangelo D, Tinaburri L and Dellambra E (2017) The role of p16inK4a pathway in human epidermal stem cell self-renewal, aging and cancer. Int J Mol Sci 18:1591. ). P16 is found to be mutated or deleted in different types of cancer (Yang Z et al., 2016Yang Z, Hu J, Li D and Pan X (2016) Adenovirus with p16 gene exerts antitumor effect on laryngeal carcinoma Hep2 cells. Mol Med Rep 14:1425-1429. ) and the restoration of its expression mediated by adenovirus (Ad-P16) resulted in antitumor effect in different tumor cell lines with functional pRb protein, but none or reduced activity in cell lines with mutated or null pRb (Grim et al., 1997Grim J, D'Amico A, Frizelle S, Zhou J, Kratzke RA and Curiel DT (1997) Adenovirus-mediated delivery of p16 to p16-deficient human bladder cancer cells confers chemoresistance to cisplatin and paclitaxel. Clin Cancer Res 3:2415-2423; Craig et al., 1998Craig C, Kim M, Ohri E, Wersto R, Katayose D, Li Z, Choi YH, Mudahar B, Srivastava S, Seth P et al. (1998) Effects of adenovirus-mediated p16 INK4A expression on cell cycle arrest are determined by endogenous p16 and Rb status in human cancer cells. Oncogene 16:265-72.; Campbell et al., 2000Campbell I, Magliocco A, Moyana T, Zheng C and Xiang J (2000) Adenovirus-mediated p16 INK4 gene transfer significantly suppresses human breast cancer growth. Cancer Gene Ther 7:1270-8. ) (^{Table S2} Table S2 - Adenoviral vectors inducing growth suppressors. ). Ad-P16 also increased radiotherapy efficiency in head and neck cancer (Rhee et al., 2003Rhee JG, Li DQ, O’Malley BW and Suntharalingam M (2003) Combination radiation and adenovirus-mediated p16INK4A gene therapy in a murine model for head and neck cancer. ORL J Otohinolaryngol Relat Spec 65:144-154. ) but conferred chemoresistance to cisplatin and paclitaxel in a P16-negative bladder cancer cell line (Grim et al., 1997Grim J, D'Amico A, Frizelle S, Zhou J, Kratzke RA and Curiel DT (1997) Adenovirus-mediated delivery of p16 to p16-deficient human bladder cancer cells confers chemoresistance to cisplatin and paclitaxel. Clin Cancer Res 3:2415-2423).

Directly modulating pRb expression, it was observed that the induction of WT pRb only had an antitumor effect in cell lines that lost the RB gene (Fueyo et al., 1998Fueyo J, Gomez-Manzano C, Yung WKA, Liu T-J, Alemany R, Bruner JM, Chintala SK, Rao JS, Levin VA and Kyritsis AP (1998) Suppression of human glioma growth by adenovirus-mediated Rb gene transfer. Neurology 50:1307-1315. ) or in a heterozygous RB (+/-) mouse (Riley et al., 1996Riley DJ, Nikitin AYu and Lee WH (1996) Adenovirus-mediated retinoblastoma gene therapy suppresses spontaneous pituitary melanotroph tumors in Rb(+/-) mice. Nat Med 2:1316-1321. ), but had no relevant effect in cervical cancer cells with inactivated pRb caused by Human Papillomavirus (HPV) infection (Ip et al., 2001Ip SM, Huang TG, Yeung WSB and Ngan HYS (2001) pRb-expressing adenovirus Ad5-Rb attenuates the p53-induced apoptosis in cervical cancer cell lines. Eur J Cancer 37:2475-2483. ). On the other hand, the adenoviral induction of a hypo-phosphorylated pRb variant resulted in tumor suppression in WT pRb cell lines (Roig et al., 2004Roig JM, Molina MA, Cascante A, Calbó J, Carbó N, Wirtz U, Sreedharan S, Fillat C and Mazo A (2004) Adenovirus-Mediated Retinoblastoma 94 Gene Transfer Induces Human Pancreatic Tumor Regression in a Mouse Xenograft Model. Clin Cancer Res 10:1454-1462.), demonstrating that pRb-based therapy should consider not just the presence but also functionality of pRb in the tumor.

Besides P16, the same gene locus INK4A/ARF also encodes P14ARF, another tumor suppressor that leads to cell cycle arrest and indirectly promotes p53 activation (Deng et al., 2002Deng X, Kim M, Vandier D, Jung Y jin, Rikiyama T, Sgagias MK, Goldsmith M and Cowan KH (2002) Recombinant adenovirus-mediated p14ARF overexpression sensitizes human breast cancer cells to cisplatin. Biochem Biophys Res Commun 296:792-798. ; Agrawal et al., 2006Agrawal A, Yang J, Murphy RF and Agrawal DK (2006) Regulation of the p14ARF-Mdm2-p53 pathway: An overview in breast cancer. Exp Mol Pathol 81:115-122. ). The induction of P14ARF expression by adenoviral vectors (Ad-p14ARF) demonstrated promising results, but the presence of the TP53 WT gene appears to be essential for its higher antitumor efficacy (Yang et al., 2000Yang CT, You L, Yeh CC, Chang JWC, Zhang F, McCormick F and Jablons DM (2000) Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells. J Natl Cancer Inst 92:636-641. ; Deng et al., 2002Deng X, Kim M, Vandier D, Jung Y jin, Rikiyama T, Sgagias MK, Goldsmith M and Cowan KH (2002) Recombinant adenovirus-mediated p14ARF overexpression sensitizes human breast cancer cells to cisplatin. Biochem Biophys Res Commun 296:792-798. ; Kim et al., 2004Kim M, Sgagias M, Deng X, Jung YJ, Rikiyama T, Lee K, Ouellette M and Cowan K (2004) Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression. Biochem Biophys Res Commun 320:138-144. ). The combination of Ad-p14ARF with an adenovirus expressing p53 synergistically increased the cytotoxic effect even in null TP53 cell lines (Lu et al., 2002Lu W, Lin J and Chen J (2002) Expression of p14ARF overcomes tumor resistance to p53. Cancer Res 62:1305-1310.; Tango et al., 2002Tango Y, Fujiwara T, Itoshima T, Takata Y, Katsuda K, Uno F, Ohtani S, Tani T, Roth JA and Tanaka N (2002) Adenovirus-mediated p14ARF gene transfer cooperates with Ad5CMV-p53 to induce apoptosis in human cancer cells. Hum Gene Ther 13:1373-1382. ), indicating that this strategy may be a good alternative for tumors lacking p53.

Interestingly, another relevant pathway altered in cancer is the Janus kinases/signal transducer and activation of transcription (JAK/STAT). It is activated by cytokines and can control immune signaling, growth, apoptosis, tissue repair, hematopoiesis, etc (Lin, 2010Lin YC, Lin CK, Tsai YH, Weng HH, Li YC, You L, Chen JK, Jablons DM and Yang CT (2010) Adenovirus-mediated SOCS3 gene transfer inhibits the growth and enhances the radiosensitivity of human non-small cell lung cancer cells. Oncol Rep 24:1605-1612. ; Owen et al., 2019Owen KL, Brockwell NK and Parker BS (2019) JAK-STAT Signaling: A Double-Edged Sword of Immune Regulation and Cancer Progression. Cancers 11:2002. ). STAT3 is considered an oncogene and the JAK/STAT pathway is often constitutively activated in cancer (Lin, 2010Lin YC, Lin CK, Tsai YH, Weng HH, Li YC, You L, Chen JK, Jablons DM and Yang CT (2010) Adenovirus-mediated SOCS3 gene transfer inhibits the growth and enhances the radiosensitivity of human non-small cell lung cancer cells. Oncol Rep 24:1605-1612. ). The use of adenovirus expressing suppressors of cytokine signaling (SOCS) induces a negative feedback control leading to this pathway inactivation (Liu et al., 2013bLiu L, Li W, Wei X, Cui Q, Lou W, Wang G, Hu X and Qian C (2013b) Potent antitumor activity of oncolytic adenovirus-mediated SOCS1 for hepatocellular carcinoma. Gene Ther 20:84-92. ). This strategy was effective against several types of cancer cells, in addition to improve radiosensitivity (Lin, 2010Lin YC, Lin CK, Tsai YH, Weng HH, Li YC, You L, Chen JK, Jablons DM and Yang CT (2010) Adenovirus-mediated SOCS3 gene transfer inhibits the growth and enhances the radiosensitivity of human non-small cell lung cancer cells. Oncol Rep 24:1605-1612. ; Sugase, et al., 2018Sugase T, Takahashi T, Serada S, Fujimoto M, Ohkawara T, Hiramatsu K, Nishida T, Hirota S, Saito Y, Tanaka K et al. (2018) SOCS1 gene therapy has antitumor effects in imatinib-resistant gastrointestinal stromal tumor cells through FAK/PI3 K signaling. Gastric Cancer 21:968-976. ; Liu et al., 2013bLiu L, Li W, Wei X, Cui Q, Lou W, Wang G, Hu X and Qian C (2013b) Potent antitumor activity of oncolytic adenovirus-mediated SOCS1 for hepatocellular carcinoma. Gene Ther 20:84-92. ).

MYC is another important gene found frequently altered in cancer (Dang,2012Dang CV (2012) MYC on the path to cancer. Cell 149:22-35. ), which participates in cell growth regulation (Stine et al., 2015Stine ZE, Walton ZE, Altman BJ, Hsieh AL and Dang CV (2015) MYC, metabolism, and cancer. Cancer Discov 5:1024-1039. ). In tumors, MYC is usually amplified, leading to its constitutive activation (Stine et al., 2015). Different strategies have been developed for MYC inhibition, such as adenovirus expressing antisense c-MYC (Chen et al., 2001Chen JP, Lin C, Xu CP, Zhang XY, Fu M, Deng YP, Wei Y and Wu M (2001) Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo. J Gastroenterol Hepatol (Australia) 16:22-28. ; Xie et al., 2009Xie XK, Yang DS, Ye ZM and Tao HM (2009) Enhancement effect of adenovirus-mediated antisense c-myc and caffeine on the cytotoxicity of cisplatin in osteosarcoma cell lines. Chemotherapy 55:433-440. ) or shRNA anti-MYC (Li Y et al., 2013Li Y, Zhang B, Zhang H, Zhu X, Feng D, Zhang D, Zhuo B, Li L and Zheng J (2013) Oncolytic adenovirus armed with shRNA targeting MYCN gene inhibits neuroblastoma cell proliferation and in vivo xenograft tumor growth. J Cancer Res Clin Oncol 139:933-941. ) leading to tumor inhibition in vivo and in vitro (^{Table S1} Table S1 - Adenoviral vectors modulating proliferation and survival of tumor cells. ). Other targets involved in cell proliferation and survival employed in adenovirus gene therapy include survivin inhibition (Fei et al., 2008Fei Q, Zhang H, Fu L, Dai X, Gao B, Ni M, Ge C, Li J, Ding X, Ke Y et al. (2008) Experimental cancer gene therapy by multiple anti-survivin hammerhead ribozymes. Acta Biochimica et Biophysica Sinica 40:466-477. ; Shen et al., 2009Shen W, Wang CY, Wang XH and Fu ZX (2009) Oncolytic adenovirus mediated Survivin knockdown by RNA interference suppresses human colorectal carcinoma growth in vitro and in vivo. J Exp Clin Cancer Res 28:81. ), Ki-67 silencing (Zheng et al., 2009Zheng JN, Pei DS, Mao LJ, Liu XY, Mei DD, Zhang BF, Shi Z, Wen RM and Sun XQ (2009) Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encoding mRNA. Cancer Gene Ther 16:20-32. ; Liu J et al., 2012Liu Z, Li J, Li J, Huang J, Ke F, Qi Q, Jiang X and Zhong Z (2012) Mannan-modified Ad5-PTEN treatment combined with docetaxel improves the therapeutic effect in H22 tumor-bearing mice. Int J Nanomedicine 7:5039-5049. ), and epidermal growth factor receptor (EGFR) expression (Yan et al., 2020Yan M, Chen J, Jiang H, Xie Y, Li C, Chen L, Yang B and Cao J (2020) Effective inhibition of cancer cells by recombinant adenovirus expressing EGFRtargeting artificial microRNA and reversedcaspase-3. PloS One 15:e0237098.).

These data suggest that it is important to take advantage of altered genes that are contributing to the uncontrolled proliferation and survival phenotype. One main problem is that the same therapy is not necessarily effective against tumors harboring different alterations of a pathway or even mutations of the same gene. In this case, the status of the target gene should always be considered.

Inducing tumor cell death and suicide gene therapy

Evading cell death is an important tumor hallmark and loss of death regulators is frequent in cancer (Hanahan and Weinberg, 2011Hanahan D and Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell 144:646-674. ). Several studies have focused on restoring death activators in an attempt to induce tumor cell death. Adenovirus expressing tumor necrosis factor receptor superfamily member 6 (FAS) ligand (Ad-FASL) contributed to cell death induction in different types of tumors (Zheng et al., 2005Zheng S-Y, Li D-C, Zhang Z-D, Zhao J and Ge J-F (2005) Adenovirus-mediated FasL gene transfer into human gastric carcinoma. World J Gastroenterol 11:3446-3450.; Sudarshan et al., 2005Sudarshan S, Holman DH, Hyer ML, Voelkel-Johnson C, Dong JY and Norris JS (2005) In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer. Cancer Gene Ther 12:12-18. ; ElOjeimy et al., 2006ElOjeimy S, McKillop JC, El-Zawahry AM, Holman DH, Liu X, Schwartz DA, Day TA, Dong JY and Norris JS (2006) FasL gene therapy: A new therapeutic modality for head and neck cancer. Cancer Gene Ther 13:739-745. ). Interestingly, the expression of caspase or pro-caspase 3 mediated by adenovirus did not have an effect on apoptosis induction in glioma (Shinoura et al., 2000Shinoura N, Muramatsu Y, Yoshida Y, Asai A, Kirino T and Hamada H (2000) Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells. Exp Cell Res 256:423-433), liver (Yamabe et al., 1999Yamabe K, Shimizu S, Ito T, Yoshioka Y, Nomura M, Narita M, Saito I, Kanegae Y and Matsuda H (1999) Cancer gene therapy using a pro-apoptotic gene, caspase-3. Gene Ther 6:1952-1959.) and prostate cancer (Li et al., 2001Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, Fang B, Denner L and Marcelli M (2001) Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 61:186-191.). High death rates were only achieved in combination with Ad-FASL (Shinoura et al., 2000Shinoura N, Muramatsu Y, Yoshida Y, Asai A, Kirino T and Hamada H (2000) Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells. Exp Cell Res 256:423-433) or the chemotherapy etoposide as a death stimulus (Yamabe et al., 1999Yamabe K, Shimizu S, Ito T, Yoshioka Y, Nomura M, Narita M, Saito I, Kanegae Y and Matsuda H (1999) Cancer gene therapy using a pro-apoptotic gene, caspase-3. Gene Ther 6:1952-1959.). In contrast, pro-caspase 7 induction resulted in cell death but only in two of five cell lines tested (Li et al., 2001Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, Fang B, Denner L and Marcelli M (2001) Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 61:186-191.). Overexpression of B-Cell CLL/Lymphoma 2 (BCL-2) pro-apoptotic family members such as BCL2 Antagonist/Killer 1 (BAK) and BCL2 Associated X (BAX) also demonstrated effective antitumor capacity through apoptosis induction (^{Table S3} Table S3 - Adenoviral vectors inducing cell death. ) and improvement of radio- and chemotherapy sensibility after Ad administration (Arafat et al., 2000Arafat WO, Gómez-Navarro J, Xiang J, Barnes MN, Mahasreshti P, Alvarez RD, Siegal GP, Badib AO, Buchsbaum D, Curiel DT et al. (2000) An adenovirus encoding proapoptotic Bax induces apoptosis and enhances the radiation effect in human ovarian cancer. Mol Ther 1:545-554. ; Tsuruta et al., 2001Tsuruta Y, Mandai M, Konishi I, Kuroda H, Kusakari T, Yura Y, Hamid AA, Tamura I, Kariya M and Fujii S (2001) Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines. Eur J Cancer 37:531-541. ). Nonetheless, Ad-BAK was not able to induce cell death in a breast caspase-3 deficient-cell line (Pataer et al., 2000Pataer A, Fang B, Yu R, Kagawa S, Hunt KK, McDonnell TJ, Roth JA and Swisher SG (2000) Adenoviral Bak overexpression mediates caspase-dependent tumor killing. Cancer Res 60:788-792.). Exploring tumor specificity, Ad-BAX under control of vascular endothelial growth factor (VEGF) promoter conferred a higher antitumor effect under hypoxic conditions in lung cancer (Kaliberov et al., 2002Kaliberov SA, Buchsbaum DJ, Gillespie GY, Curiel DT , Arafat WO, Carpenter M and Stackhouse MA (2002) Adenovirus-mediated transfer of BAX driven by the vascular endothelial growth factor promoter induces apoptosis in lung cancer cells. Mol Ther 6:190-198. ). In prostate (Lowe et al., 2001Lowe SL, Rubinchik S, Honda T, Mcdonnell TJ, Dong J-Y and Norris JS (2001) Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells. Gene Ther 8:1363-1371.) and ovarian cancer (Tai et al., 1999Tai YT, Strobel T, Kufe D and Cannistra SA (1999) In vivo cytotoxicity of ovarian cancer cells through tumor-selective expression of the BAX gene. Cancer Res 59:2121-2126.), Ad-BAX under control of specific promoters conferred specificity and high cytotoxicity. The effect of BAX expression was also studied in combination with IL-24 (Li et al., 2010Li J, Shi L, Zhang X, Kang X, Wen Y, Qian H, Zhou Y, Xu W, Zhang Y, Wu M et al. (2010) Recombinant adenovirus IL-24-Bax promotes apoptosis of hepatocellular carcinoma cells in vitro and in vivo. Cancer Gene Ther 17:771-779. ) through an adenovirus expressing the TNF-related apoptosis-inducing ligand (TRAIL) and with the chemotherapeutic agent Gemcitabine (Wack et al., 2008Wack S, Rejiba S, Parmentier C, Aprahamian M and Hajri A (2008) Telomerase transcriptional targeting of inducible bax/TRAIL gene therapy improves gemcitabine treatment of pancreatic cancer. Mol Ther 16:252-260. ). In all cases, the antitumor effect was improved synergistically.

P53 is another important protein that controls cell apoptosis, inducing cell death in response to stressful stimuli, besides several other processes related to tumor suppression. P53 is the tumor suppressor most frequently mutated in cancer (Bieging et al., 2014Bieging KT, Mello SS and Attardi LD (2014) Unravelling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer 14:359-370. ) and its restoration has been extensively used in gene therapy mediated by adenovirus (Ad-P53) in different types of cancer (reviewed before by Tazawa et al., 2013Tazawa H, Kagawa S and Fujiwara T (2013) Advances in adenovirus-mediated p53 cancer gene therapy. Exp Opin Biol Ther 13:1569-1583. ). In prostate cancer, for example, several studies have employed Ad-P53 gene therapy and showed antitumor activity (Tamura et al., 2018Tamura RE, de Luna IV, Lana MG and Strauss BE (2018) Improving adenoviral vectors and strategies for prostate cancer gene therapy. Clinics 73:e476s ). Adenovirus expressing P53 under control of a P53-responsive promoter demonstrated effective tumor suppression (Tamura et al., 2016Tamura RE, da Silva Soares RB, Costanzi-Strauss E and Strauss BE (2016) Autoregulated expression of p53 from an adenoviral vector confers superior tumor inhibition in a model of prostate carcinoma gene therapy. Cancer Biol Ther 17:1221-1230.), which is potentialized when the arginine-glycine-aspartic acid (RGD) motif is incorporated in the adenoviral fiber protein, leading to a higher tumor cell death effect (Tamura et al., 2017Tamura RE, Hunger A, Fernandes DC, Laurindo FR, Costanzi-Strauss E and Strauss BE (2017) Induction of oxidants distinguishes susceptibility of prostate carcinoma cell lines to p53 gene transfer mediated by an improved adenoviral vector. Hum Gene Ther 28:639-653. ) and higher chemotherapy sensitivity (Tamura et al., 2020). Tamura RE, Lana MG, Costanzi-Strauss E and Strauss BE (2020) Combination of cabazitaxel and p53 gene therapy abolishes prostate carcinoma tumor growth. Gene Ther 27:15-26. In colon cancer, the same strategy, Ad-P53 containing RGD and P53-responsive promoter, was only effective in P53 WT or null cell lines and in a mutant TP53 tumor cell, the combination with IFNβ was necessary to induce cell death (Del Valle et al., 2021Del Valle PR, Mendonça SA, Antunes F, Hunger A, Tamura RE, Zanatta DB and Strauss BE (2021) Exploration of p53 plus interferon-beta gene transfer for the sensitization of human colorectal cancer cell lines to cell death. Cancer Biol Ther 22:301-310. ). Several clinical trials employed adenovirus-expressing P53 for treating different types of cancer and for safety confirmation (See on clinicaltrials.govClinicalTrials.gov, gov, https://clinicaltrials.gov/ (accessed 21 June 2022).
https://clinicaltrials.gov/... ). Currently in 2002, a phase II clinical trial is combining Ad-P53 with an approved immune checkpoint inhibitor in a cohort of 40 head and neck cancer patients and other tumors.

Such studies resulted in Gendicine® (Shenzhen SiBiono GeneTech, Guangdong, China), an adenoviral p53 gene therapy approved in China for treating Head and neck cancers in 2003. It was also the first approved gene therapy drug, which confers higher survival rates and improved treatment compared to conventional therapies (radio and chemotherapy) with no severe side effects (Zhang et al., 2018aZhang WW, Li L, Li D, Liu J, Li X, Li W, Xu X, Zhang MJ, Chandler LA, Lin H et al. (2018a) The First Approved Gene Therapy Product for Cancer Ad-p53 (Gendicine): 12 Years in the Clinic. Hum Gene Ther 29:160-179. ). Besides head and neck, gendicine can also be used for treating lung, ovarian, liver and other cancers (Zhang et al., 2018aZhang WW, Li L, Li D, Liu J, Li X, Li W, Xu X, Zhang MJ, Chandler LA, Lin H et al. (2018a) The First Approved Gene Therapy Product for Cancer Ad-p53 (Gendicine): 12 Years in the Clinic. Hum Gene Ther 29:160-179. ). Other two adenoviral vectors expressing p53 have been investigated by pharmaceutical companies, Advexin® (Introgen Therapeutics, Multivir, Inc, both of Houston, TX) and SCH58500 (Merck & Co, Schering-Plough, Kenilworth, NJ).

Downstream targets of P53 have been investigated as well, including adenoviral vectors expressing P53 upregulated modulator of apoptosis (PUMA) and NADPH oxidase activator (NOXA), other two members of the BCL-2 family that participate in P53-mediated apoptosis (Agrawal et al., 2006Agrawal A, Yang J, Murphy RF and Agrawal DK (2006) Regulation of the p14ARF-Mdm2-p53 pathway: An overview in breast cancer. Exp Mol Pathol 81:115-122. ; Elmore, 2007Elmore S (2007) Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol 35:495-516. ), in different tumor types (^{Table S3} Table S3 - Adenoviral vectors inducing cell death. ).

A different method for inducing cell death is through the expression of suicide genes in tumor cells (Düzgüneş, 2019Düzgüneş N (2019) Origins of suicide gene therapy. Suicide Gene Therapy: Methods and Protocols. Methods Mol Biol 1895:1-9). The best described is the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system, in which HSVtk converts the prodrug ganciclovir into a nucleoside analog consequently occasioning cell cycle arrest and cell death (Beltinger et al., 1999Beltinger C, Fulda S, Kammertoens T, Meyer E, Uckert W and Debatin KM (1999) Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. Proc Natl Acad Sci U S A 96:8699-8704). The use of adenovirus carrying HSVtk in tumor cells has shown a relevant antitumor effect with high cytotoxicity to ganciclovir in a variety of pre- clinical trials (^{Table S3} Table S3 - Adenoviral vectors inducing cell death. ). Clinical trials using adenovirus-expressing HSVtk in combination with GCV demonstrated its safety and efficacy in liver cancer (Sangro et al., 2010Sangro B, Mazzolini G, Ruiz M, Ruiz J, Quiroga J, Herrero I, Qian C, Benito A, Larrache J, Olagüe C et al. (2010) A phase I clinical trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma. Cancer Gene Ther 17:837-843. ) and glioma patients in combination with radio- and chemotherapy (Chiocca et al., 2011Chiocca EA, Aguilar LK, Bell SD, Kaur B, Hardcastle J, Cavaliere R, McGregor J, Lo S, Ray-Chaudhuri A, Chakravarti A et al. (2011) Phase IB study of gene-mediated cytotoxic immunotherapy adjuvant to up-front surgery and intensive timing radiation for malignant glioma. J Clin Oncol 29:3611-3619. ; Ji et al., 2016Ji N, Weng D, Liu C, Gu Z, Chen S, Guo Y, Fan Z, Wang X, Chen J, Zhao Y et al. (2016) Adenovirus-mediated delivery of herpes simplex virus thymidine kinase administration improves outcome of recurrent high-grade glioma. Oncotarget7:4369-4378. ). The system cytosine deaminase /5’-Fluorocytosine (CD/5’-FC) was also explored in adenovirus gene therapy. In this case, CD converts the prodrug 5’-FC into a toxic molecule (Düzgüneş, 2019). The combination between the systems TK and CD carried to tumor cells by adenoviruses led to a synergistic antitumor effect in gastric cancer (Luo et al., 2012Luo XR, Li JS, Niu Y and Miao L (2012) Adenovirus-mediated double suicide gene selectively kills gastric cancer cells. Asian Pac J Cancer Prev 13:781-784.). In pancreatic cancer preclinical studies, AdHSVTk/CD increased the radiotherapy effect (Freytag et al., 2007Freytag SO, Barton KN, Brown SL, Narra V, Zhang Y, Tyson D, Nall C, Lu M, Ajlouni M, Movsas B et al. (2007) Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer. Mol Ther 15:1600-1606. ) and a phase I clinical trial demonstrated tolerability in combination with gemcitabine chemotherapy (Lee et al., 2020Lee JC, Shin DW, Park H, Kim J, Youn Y, Kim JH, Kim J and Hwang JH (2020) Tolerability and safety of EUS-injected adenovirus-mediated double-suicide gene therapy with chemotherapy in locally advanced pancreatic cancer: a phase 1 trial. Gastrointest Endosc 92:1044-1052.e1. ). HSVtk/GCV and CD/5’-FC in prostate cancer also appeared to be safe in phase I clinical trials (Freytag et al., 2003Freytag SO, Stricker H, Pegg J, Paielli D, Pradhan DG, Peabody J, Deperalta-Venturina M, Xia X, Brown S, Lu M et al. (2003) Phase I Study of Replication-Competent Adenovirus-Mediated Double-Suicide Gene Therapy in Combination with Conventional-Dose Three-Dimensional Conformal Radiation Therapy for the Treatment of Newly Diagnosed, Intermediate- to High-Risk Prostate Cancer. Cancer Res 63:7497-506; Barton et al., 2008Barton KN, Stricker H, Brown SL, Elshaikh M, Aref I, Lu M, Pegg J, Zhang Y, Karvelis KC, Siddiqui F et al. (2008) Phase I study of noninvasive imaging of adenovirus-mediated gene expression in the human prostate. Mol Ther 16:1761-1769. ).

Restoring the expression of tumor suppressors or inducing cell death by different means is essential in any strategy to destroy the tumoral cell mass. Therefore, it is natural that the first available product and several clinical trials assays are intended to promote direct tumor cell death and recruitment of the immune system to eliminate any remaining cells.

Inhibiting angiogenesis

Angiogenesis is the construction of new blood vessels coming from pre-existing vessels, which is induced by tumor cells signaling and essential for tumor growth and metastasis dissemination (Chen et al., 2000Chen CT, Lin J, Li Q, Phipps SS, Jakubczak JL, Stewart DA, Skripchenko Y, Forry-Schaudies S, Wood J, Schnell C et al. (2000) Antiangiogenic Gene Therapy for Cancer via Systemic Administration of Adenoviral Vectors Expressing Secretable Endostatin. Hum Gene Ther 11:1983-96.). The most utilized anti-angiogenic proteins in gene therapy are statins as endostatin and angiostatin (Chen et al., 2000Chen CT, Lin J, Li Q, Phipps SS, Jakubczak JL, Stewart DA, Skripchenko Y, Forry-Schaudies S, Wood J, Schnell C et al. (2000) Antiangiogenic Gene Therapy for Cancer via Systemic Administration of Adenoviral Vectors Expressing Secretable Endostatin. Hum Gene Ther 11:1983-96.). Both molecules are natural fragments of larger proteins (endostatin from XVIII collagen and angiostatin from plasminogen) and their anti-angiogenic capability may be due to VEGF downregulation, a well-known molecule responsible for angiogenesis induction (Hajitou et al., 2002Hajitou A, Grignet C, Devy L, Berndt S, Blacher S, Deroanne CF, Bajou K, Fong T, Chiang Y, Foidart JM et al. (2002) The antitumoral effect of endostatin and angiostatin is associated with a down-regulation of vascular endothelial growth factor expression in tumor cells. FASEB J 16:1802-1804. ). Different studies evaluated the antitumoral ability of adenovirus expressing endostatin, angiostatin, and different fragments of plasminogen, demonstrating to be effective against the angiogenic phenotype of endothelial cells in vitro and tumor suppression in vivo, influencing mainly tumor vessels formation, cell migration, invasion, and metastasis (^{Table S4} Table S4 - Adenoviral vectors modulating tumoral angiogenesis. ).

Several mechanisms using adenovirus were proposed to decrease VEGF expression in cancer: antisense-VEGF (Im et al., 2001Im SA, Kim JS, Gomez-Manzano C, Fueyo J, Liu TJ, Cho MS, Seong CM, Lee SN, Hong YK and Yung WKA (2001) Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF. Br J Cancer 84:1252-1257. ), soluble forms of VEGF receptor (VEGFR/Flt-1 or VEGFR2/Flk-1) (Kong et al., 1998Kong HL, Hecht D, Song W, Kovesdi I, Hackett NR, Yayon A and Crystal RG (1998) Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Hum Gene Ther 9:823-833. ; Takayama et al., 2000Takayama K, Ueno H, Nakanishi Y, Sakamoto T, Inoue K, Shimizu K, Oohashi H and Hara N (2000) Suppression of Tumor Angiogenesis and Growth by Gene Transfer of a Soluble Form of Vascular Endothelial Growth Factor Receptor into a Remote Organ. Cancer Res 60:2169-2177.; Hoshida et al., 2002Hoshida T, Sunamura M, Duda DG, Egawa S, Miyazaki S, Shineha R, Hamada H, Ohtani H, Satomi S and Matsuno S (2002) Gene Therapy for Pancreatic Cancer Using an Adenovirus Vector Encoding Soluble flt-1 Vascular Endothelial Growth Factor Receptor. Pancreas25:111-121.; Yoshimura et al., 2004Yoshimura I, Mizuguchi Y, Miyajima A, Asano T, Tadakuma T and Hayakawa M (2004) Suppression of lung metastasis of renal cell carcinoma by the intramuscular gene transfer of a soluble form of vascular endothelial growth factor receptor I. J Urol 171:2467-2470. ; Schmitz et al., 2005Schmitz V, Kornek M, Hilbert T, Dzienisowicz C, Raskopf E, Rabe C, Sauerbruch T, Qian C and Caselmann WH (2005) Treatment of metastatic colorectal carcinomas by systemic inhibition of vascular endothelial growth factor signaling in mice. World J Gastroenterol 11:4332-4336.; Wu et al., 2006Wu Y, Li Z-Y, Zhao X, Kan B and Wei Y-Q (2006) Inhibition of Ovarian Tumor Growth by Gene Therapy with Recombinant Soluble Vascular Endothelial Growth Factor Receptor 2. Hum Gene Ther 17:941-948.) and the vascular endothelial growth inhibitor (VEGI) fused with endostatin (Pan et al., 2004Pan X, Wang Y, Zhang M, Pan W, Qi ZT and Cao GW (2004) Effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenoviruses on antiangiogenesis. World J Gastroenterol 10:1409-1414. ) are examples of molecules used in gene therapy that showed a reduction on neovascularization, increase in apoptosis and tumor suppression in vivo (^{Table S4} Table S4 - Adenoviral vectors modulating tumoral angiogenesis. ).

In addition, hepatocellular growth factor (HGF) plays a role in tumor malignant phenotype (Saimura et al., 2002Saimura M, Nagai E, Mizumoto K, Maehara N, Okino H, Katano M, Matsumoto K, Nakamura T, Narumi K, Nukiwa T et al. (2002) Intraperitoneal injection of adenovirus-mediated NK4 gene suppresses peritoneal dissemination of pancreatic cancer cell line AsPC-1 in nude mice. Cancer Gene Ther 9:799-806. ). Several studies using Ad carrying its antagonist Nk4 showed anti-proliferative and anti-angiogenic activity in different types of cancer (^{Table S4} Table S4 - Adenoviral vectors modulating tumoral angiogenesis. ). Other examples of adenoviral gene therapy focusing on antiangiogenic mechanisms includes the expression of pigment epithelium-derived factor (PEDF) (Mahtabifard et al., 2003Mahtabifard A, Merritt RE, Yamada RE, Crystal RG and Korst RJ (2003) In vivo gene transfer of pigment epithelium-derived factor inhibits tumor growth in syngeneic murine models of thoracic malignancies. J Tho Cardiovas Surg 126:28-38. ; Wang et al., 2003Wang L, Schmitz V, Perez-Mediavilla A, Izal I, Prieto J and Qian C (2003) Suppression of angiogenesis and tumor growth by adenoviral-mediated gene transfer of pigment epithelium-derived factor. Mol Ther 8:72-79. ; Merritt et al., 2004Merritt RE, Yamada RE, Wasif N, Crystal RG and Korst RJ (2004) Effect of inhibition of multiple steps of angiogenesis in syngeneic murine pleural mesothelioma. Ann Thorac Surg 78:1042-1051. ; Guan et al., 2007Guan M, Jiang H, Xu C, Xu R, Chen Z and Lu Y (2007) Adenovirus-mediated PEDF expression inhibits prostate cancer cell growth and results in augmented expression of PAI-2. Cancer Biol Ther 6:419-425. ); endothelium-specific receptor tyrosine kinase (Tie2) (Lin et al., 1998Lin P, Buxton JA, Acheson A, Radziejewski C, Maisonpierre PC, Yancopoulos GD, Channon KM, Hale LP, Dewhirst MW, George SE et al. (1998) Antiangiogenic gene therapy targeting the endothelium-specific receptor tyrosine kinase Tie2. Proc Natl Acad Sci U S A 95:8829-8834; Popkov et al., 2005 Popkov M, Jendreyko N, McGavern DB, Rader C, Barbas CF 3rd (2005) Targeting Tumor Angiogenesis with Adenovirus-Delivered Anti-Tie-2 Intrabody. Cancer Res 65:972-981.); fragments and alterations of thrombospondin 1 (Liu et al., 2003Liu P, Wang Y, Li YH, Yang C, Zhou YL, Li B, Lu SH, Yang RC, Cai YL, Tobelem G et al. (2003) Adenovirus-mediated gene therapy with an antiangiogenic fragment of thrombospondin-1 inhibits human leukemia xenograft growth in nude mice. Leuk Res 27:701-708. ); angiotensinogen (Bouquet et al., 2006Bouquet C, Lamandé N, Brand M, Gasc JM, Jullienne B, Faure G, Griscelli F, Opolon P, Connault E, Perricaudet M et al. (2006) Suppression of angiogenesis, tumor growth, and metastasis by adenovirus-mediated gene transfer of human angiotensinogen. Mol Ther 14:175-182. ); human 16k PRL (Nguyen et al., 2007Nguyen NQN, Cornet A, Blacher S, Tabruyn SP, Foidart JM, Noël A, Martial JA and Struman I (2007) Inhibition of tumor growth and metastasis establishment by adenovirus-mediated gene transfer delivery of the antiangiogenic factor 16K hPRL. Mol Ther 15:2094-2100. ); amino-terminal fragment of urokinase (ATF) (Li et al., 1999Li H, Griscelli F, Lindenmeyer F, Opolon P, Sun L-Q, Connault E, Soria J, Soria C, Perricaudet M, Yeh P et al. (1999) Systemic Delivery of Antiangiogenic Adenovirus AdmATF Induces Liver Resistance to Metastasis and Prolongs Survival of Mice. Hum Gene Ther 10:3045-53); fibroblast growth factor receptor (FGFR) (Compagni et al., 2000Compagni A, Wilgenbus P, Impagnatiello M-A, Cotten M and Christofori G (2000) Fibroblast Growth Factors Are Required for Efficient Tumor Angiogenesis. Cancer Res 60:7163-7169. ) and platelet factor 4 (PF4) (Tanaka et al., 1997Tanaka T, Manome Y, Wen P, Kufe DW and Fine HA (1997) Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nat Med 3:437-442. ).

All mechanisms mentioned above indicated that gene therapy using antiangiogenic molecules provides high anticancer efficacy in pre-clinical assays, resulting in tumor growth suppression in almost all cell lines and in vivo models studied. It is also important to note that this strategy does not depend on a specific mutation or is restricted to a specific type of cancer. The expression of pro-angiogenic factors and stimulation of tumor blood vessel formation are frequently found in cancer, being an interesting target for cancer treatment of solid tumors.

Focusing on invasion and metastasis

The tumor malignant phenotype is also characterized by adjacent tissue invasion and metastasis to distant sites (Jiang et al., 2015aJiang WG, Sanders AJ, Katoh M, Ungefroren H, Gieseler F, Prince M, Thompson SK, Zollo M, Spano D, Dhawan P et al. (2015a) Tissue invasion and metastasis: Molecular, biological and clinical perspectives. Semin Cancer Biol 35:S244-S275. ). These mechanisms are regulated mainly by the degradation of molecules responsible for cell to cell and cell to matrix adhesion, stimulus of cell migration, and through epithelium-mesenchymal transition (EMT) (Jiang et al., 2015bJiang Y-Q, Zhang Z, Cai H-R and Zhou H (2015b) Killing effect of TNF-mediated by conditionally replicating adenovirus on esophageal cancer and lung cancer cell lines. Int J Clin Exp Pathol 8:13785-13794 ). Adenoviruses expressing extracellular matrix (ECM) compounds like connexin 43 (Cx43) (Liu et al., 2015Liu D, Zhou H, Wu J, Liu W, Li Y, Shi G, Yue X, Sun X, Zhao Y, Hu X et al. (2015) Infection by Cx43 adenovirus increased chemotherapy sensitivity in human gastric cancer BGC-823 cells: Not involving in induction of cell apoptosis. Gene 574:217-224. ) or downregulating CD44 via short hairpin (sh) RNA (Lee et al., 2017Lee SY, Kim KA, Kim CH, Kim YJ, Lee JH and Kim HR (2017) CD44-shRNA recombinant adenovirus inhibits cell proliferation, invasion, and migration, and promotes apoptosis in HCT116 colon cancer cells. Int J Oncol 50:329-336. ) contributed to the reduction in invasiveness capability in cancer cells in vitro (^{Table S5} Table S5 - Adenoviral vectors modulating invasion and metastasis. ). Additionally, several studies evaluated different mechanisms to inhibit matrix metalloproteinases (MMPs) that are responsible for ECM degradation. Using adenovirus expressing siRNA against MMP2 (Chetty et al., 2006Chetty C, Bhoopathi P, Joseph P, Chittivelu S, Rao JS and Lakka S (2006) Adenovirus-mediated small interfering RNA against matrix metalloproteinase-2 suppresses tumor growth and lung metastasis in mice. Mol Cancer Ther 5:2289-2299. ; Tsung et al., 2008Tsung AJ, Kargiotis O, Chetty C, Lakka SS, Gujrati M, Spomar DG, Dinh DH and Rao JS (2008) Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model. Int J Oncol 32:557-564. ) or a ribozyme against MMP-13 mRNA (Ala-Aho et al., 2004Ala-Aho R, Ahonen M, George SJ, Heikkilä J, Grénman R, Kallajoki M and Kähäri VM (2004) Targeted inhibition of human collagenase-3 (MMP-13) expression inhibits squamous cell carcinoma growth in vivo. Oncogene 23:5111-5123. ) resulted in its downregulation in tumor cells, consequently leading to reduced invasion and migration. Natural inhibitors of MMPs, like tissue inhibitors of MMPs (TIMPs), were also explored. Adenovirus expressing TIMP-1, -2, or -3 demonstrated high antitumor effect mainly by reducing angiogenesis, invasion, and metastasis (^{Table S5} Table S5 - Adenoviral vectors modulating invasion and metastasis. ). Other different methods for MMPs inhibition include the expression of cystatin C (Kopitz et al., 2005Kopitz C, Anton M, Gansbacher B and Krüger A (2005) Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host. Cancer Res 65:8608-8612. ) and the urokinase plasminogen activator receptor (uPAR) (Lakka et al., 2001Lakka SS, Rajagopal R, Rajan MK, Mohan PM, Adachi Y, Dinh DH, Olivero WC, Gujrati M, Ali-Osman F, Roth JA et al. (2001) Adenovirus-mediated Antisense Urokinase-Type Plasminogen Activator Receptor Gene Transfer Reduces Tumor Cell Invasion and Metastasis in Non-Small Cell Lung Cancer Cell Lines. Clin Cancer Res 7:1087-1093., 2003Lakka SS, Gondi CS, Yanamandra N, Dinh DH, Olivero WC, Gujrati M and Rao JS (2003) Synergistic Down-Regulation of Urokinase Plasminogen Activator Receptor and Matrix Metalloproteinase-9 in SNB19 Glioblastoma Cells Efficiently Inhibits Glioma Cell Invasion, Angiogenesis, and Tumor Growth 1. Cancer Res 63:2454-2461; Rao et al., 2005Rao JS, Gondi C, Chetty C, Chittivelu S, Joseph PA, Lakka SS and Lakka SS (2005) Inhibition of invasion, angiogenesis, tumor growth and metastasis by adenovirus-mediated transfer of antisense uPAR and MMP-9 in non-small cell lung cancer cells. Mol Cancer Ther 4:1399-1408).

Focusing on EMT as a therapeutical target, one important protein is Mothers against decapentaplegic homolog 4 (Smad4), which is involved in cell differentiation and is found mutated in several cancers (Duda et al., 2003Duda DG, Sunamura M, Lefter LP, Furukawa T, Yokoyama T, Yatsuoka T, Abe T, Inoue H, Motoi F, Egawa SI et al. (2003) Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells. Oncogene 22:6857-6864. ; Xiao et al., 2020Xiao B, Zhang L, Liu H, Fang H, Wang C, Huang B, Liu X, Zhou X and Wang Y (2020) Oncolytic adenovirus cd55-smad4 suppresses cell proliferation, metastasis, and tumor stemness in colorectal cancer by regulating wnt/β-catenin signaling pathway. Biomedicines 8:593. ). Its overexpression mediated by an adenoviral vector in pancreatic tumor cells did not affect proliferation in vitro but resulted in tumor growth and angiogenesis inhibition in vivo (Duda et al., 2003Duda DG, Sunamura M, Lefter LP, Furukawa T, Yokoyama T, Yatsuoka T, Abe T, Inoue H, Motoi F, Egawa SI et al. (2003) Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells. Oncogene 22:6857-6864. ). In colon cancer, using oncolytic adenovirus, Smad4 expression promoted cell proliferation inhibition in vivo and in vitro, and reduced spheroids formation efficiency (Xiao et al., 2020Xiao B, Zhang L, Liu H, Fang H, Wang C, Huang B, Liu X, Zhou X and Wang Y (2020) Oncolytic adenovirus cd55-smad4 suppresses cell proliferation, metastasis, and tumor stemness in colorectal cancer by regulating wnt/β-catenin signaling pathway. Biomedicines 8:593. ).

Similar to targeting angiogenesis, invasion and metastasis are common features of cancer, seen in almost all types of tumors. Adenovirus gene therapy using key molecules involved in these processes, such as TIMPs or certain ECM compounds, seems to be another intelligent strategy for reducing the tumor malignant phenotype without limitations regarding tumor type, mutations, or alterations in important pathways that diverge among tumors.

Modulating immune signaling

Tumor cells are modulated by both adaptive and innate immune systems. Induction of inflammation may promote tumor progression by secretion of growth and survival signaling molecules, and other factors that contribute to tumor establishment. In contrast, by immune surveillance, immune cells can destroy cancer cells (Hanahan and Weinberg, 2011Hanahan D and Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell 144:646-674. ). Cancer cells can evade immune destruction in the tumor microenvironment and support pro-malignant inflammation (Hanahan and Weinberg, 2011Hanahan D and Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell 144:646-674. ).

One of the aims of adenovirus gene therapy is inducing the expression of immune components in the tumor microenvironment, such as inflammatory cytokines, that can regulate important cell pathways or activate the immunologic response, consequently triggering cell death or immune-mediated destruction (Waldmann, 2018Waldmann TA (2018) Cytokines in cancer immunotherapy. Cold Spring Harb Perspect Biol 10:a028472. ). Different cytokines are in clinical trials and some of them are already approved for cancer treatment. Although, one important implication involving cytokines in cancer therapy is the low concentration of these molecules in the tumor site and that a large quantity of systemic cytokines usually provokes high toxicity (Waldmann, 2018). The use of adenovirus to target tumor cells may be an optimist alternative to increase the efficiency of cytokine delivery in cancer and reduce systemic toxicity.

Examples of adenovirus immunotherapy include the expression of tumor necrosis factor family members (TNF), like TNFα and TRAIL. These molecules are capable of inducing tumor growth suppression when expressed in tumor cells by oncolytic or non-replicative adenovirus (^{Table S6} Table S6 - Adenoviral vectors modulating immune system. ). In some cases, oncolytic adenoviruses carrying TRAIL had a higher cytotoxic effect in comparison to virotherapy alone (Shim et al., 2010Shim SH, Lee CT, Hun Hah J, Lee JJ, Park SW, Heo DS and Sung MW (2010) Conditionally replicating adenovirus improves gene replication efficiency and anticancer effect of E1-deleted adenovirus carrying TRAIL in head and neck squamous cell carcinoma. Cancer Sci 101:482-487. ; Cao et al., 2011bCao X, Yang M, Wei RC, Zeng Y, Gu JF, Huang WD, Yang DQ, Li HL, Ding M, Wei N et al. (2011b) Cancer targeting Gene-Viro-Therapy of liver carcinoma by dual-regulated oncolytic adenovirus armed with TRAIL gene. Gene Ther 18:765-777. ; Yang et al., 2015Yang Y, Xu H, Huang W, Ding M, Xiao J, Yang D, Li H, Liu XY and Chu L (2015) Targeting lung cancer stem-like cells with TRAIL gene armed oncolytic adenovirus. J Cell Mol Med 19:915-923.; Zhou et al., 2017Zhou W, Dai S, Zhu H, Song Z, Cai Y, Lee JB, Li Z, Hu X, Fang B, He C et al. (2017) Telomerase-specific oncolytic adenovirus expressing TRAIL suppresses peritoneal dissemination of gastric cancer. Gene Ther 24:199-207. ), and improved chemotherapy treatment in bladder cancer (Mao et al., 2014Mao L, Yang C, Li L, Nai L, Fan L, Wang J, Li W, Wen R, Chen J and Zheng J (2014) Replication-competent adenovirus expressing TRAIL synergistically potentiates the antitumor effect of gemcitabine in bladder cancer cells. Tumor Biol 35:5937-5944. ). In addition, transduction of TRAIL gene to mesenchymal stem cells (MSCs) in co-culture with esophageal cancer cell lines promoted tumor cell apoptosis (Li et al., 2014Li L, Li F, Tian H, Yue W, Li S and Chen G (2014) Human mesenchymal stem cells with adenovirus-mediated TRAIL gene transduction have antitumor effects on esophageal cancer cell line Eca-109. Acta Biochim Biophys Sin 46:471-476. ).

The interferon (IFN) cytokine family is also used for cancer treatment. Induction of IFNα, -β, or -γ expression in tumor cells demonstrated a high antitumor effect in several types of cancer in vivo and in vitro (^{Table S6} Table S6 - Adenoviral vectors modulating immune system. ). Interestingly, treating cancer with Ad-IFNα resulted in higher IFNα concentration in tumors than in systemic circulation (Ohashi et al., 2005Ohashi M, Yoshida K, Kushida M, Miura Y, Ohnami S, Ikarashi Y, Kitade Y, Yoshida T and Aoki K (2005) Adenovirus-mediated interferon α gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer. Br J Cancer 93:441-449. ), and promoted regression of non-treated distant tumors as well, also inducing T-cells and natural killer cells recruitment to tumor site (Hara et al., 2007Hara H, Kobayashi A, Yoshida K, Ohashi M, Ohnami S, Uchida E, Higashihara E, Yoshida T and Aoki K (2007) Local interferon-α gene therapy elicits systemic immunity in a syngeneic pancreatic cancer model in hamster. Cancer Sci 98:455-463. ). IFNβ in an oncolytic adenovirus improved treatment (He et al., 2008He LF, Gu JF, Tang WH, Fan JK, Wei N, Zou WG, Zhang YH, Zhao LL and Liu XY (2008) Significant antitumor activity of oncolytic adenovirus expressing human interferon-β for hepatocellular carcinoma. J Gene Med 10:983-992; Park et al., 2010Park MY, Kim DR, Jung HW, Yoon HI, Lee JH and Lee CT (2010) Genetic immunotherapy of lung cancer using conditionally replicating adenovirus and adenovirus-interferon-β. Cancer Gene Ther 17:356-364. ) and adenovirus expressing IFNγ showed low systemic toxicity (Xie et al., 2013Xie FJ, Zhao P, Zhang YP, Liu FY, Nie XL, Zhu YH, Yu XM, Zheng QQ, Mao WM, Lu HY et al. (2013) Adenovirus-mediated interferon-γ gene therapy induced human pancreatic carcinoma Capan-2 cell apoptosis in vitro and in vivo. Anat Rec (Hoboken) 296:604-610. ; Zhao et al., 2007Zhao P, Zhu YH, Wu JX, Liu RY, Zhu XY, Xiao X, Li HL, Huang BJ, Xie FJ, Chen JM et al. (2007) Adenovirus-mediated delivery of human IFNγ gene inhibits prostate cancer growth. Life Sci 1:695-701. ).

Interleukins, such as IL-24, induce apoptosis and suppress growth in several tumor types (Chang et al., 2011Chang S, Yang J, Chen W, Xie Y and Sheng W (2011) Antitumor activity of an adenovirus harboring human IL-24 in colon cancer. Mol Biol Rep 38:395-401. ). Ad-IL-24 promoted tumor suppression (Chang et al., 2011Chang S, Yang J, Chen W, Xie Y and Sheng W (2011) Antitumor activity of an adenovirus harboring human IL-24 in colon cancer. Mol Biol Rep 38:395-401. ) and had its antitumor effect enhanced by radiotherapy in nasopharynx and breast cancer (Liu et al., 2013aLiu J, Zhang Y, Sun P, Xie Y, Xiang J and Yang J (2013a) Enhanced therapeutic efficacy of adenovirus-mediated interleukin-24 gene therapy combined with ionizing radiotherapy for nasopharyngeal carcinoma. Oncol Rep 30:1165-1174. ; Zhao et al., 2013Zhao Y, Li Z, Sheng W, Miao J and Yang J (2013) Radiosensitivity by ING4-IL-24 bicistronic adenovirus-mediated gene cotransfer on human breast cancer cells. Cancer Gene Ther 20:38-45. ). Furthermore, combination of IL24 and Oncostatin M (OSM) increased antitumor activity in comparison to isolated treatment in melanoma (Xu et al., 2014Xu Y, Zhang F, Qin L, Miao J, Sheng W, Xie Y, Xu X, Yang J and Qian H (2014) Enhanced in-vitro and in-vivo suppression of A375 melanoma by combined IL-24/OSM adenoviral-mediated gene therapy. Melanoma Res 24:20-27. ) and liver cancer, combining two different oncolytic adenoviruses expressing IL-24 or SOC3S resulted in higher tumor suppression when compared to alone treatments or with an empty oncolytic adenovirus (Cao et al., 2011aCao X, Wei R, Liu X, Zeng Y, Huang H, Ding M, Zhang K and Liu XY (2011a) Cancer targeting Gene-Viro-Therapy specific for liver cancer by α-fetoprotein-controlled oncolytic adenovirus expression of SOCS3 and IL-24. Acta Biochim Biophy Sin (Shangay) 43:813-821. ).

IL-12 is another important cytokine that acts as an important mediator for cancer immune destruction as it can activate NK and T cells, but it is toxic when administered systemically (Mirlekar and Pylayeva-Gupta, 2021Mirlekar B and Pylayeva-Gupta Y (2021) IL-12 family cytokines in cancer and immunotherapy. Cancers 13:167. ). Several studies using adenovirus encoding IL-12 alone demonstrated a potent antitumor effect in pre-clinical and clinical trials (reviewed before by Hernandez-Alcoceba et al., 2016Hernandez-Alcoceba R, Poutou J, Ballesteros-Briones MC and Smerdou C (2016) Gene therapy approaches against cancer using in vivo and ex vivo gene transfer of interleukin-12. Immunotherapy8:179-198. ). The combination of IL-12 oncolytic adenovirus in CIK cells in liver cancer generated higher cytotoxic effect than each separated treatment (Yang et al., 2012Yang Z, Zhang Q, Xu K, Shan J, Shen J, Liu L, Xu Y, Xia F, Bie P, Zhang X et al. (2012) Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model. PloS One 7:e44802. ) and combination with a TGFβ inhibitor in melanoma cells promoted increased antitumor immune response as well, leading to CD4+, CD8+ T and NK cells activation and IFNγ secretion in the tumor site (Jiang et al., 2017Jiang J, Zhang Y, Peng K, Wang Q, Hong X, Li H, Fan G, Zhang Z, Gong T and Sun X (2017) Combined delivery of a TGF-β inhibitor and an adenoviral vector expressing interleukin-12 potentiates cancer immunotherapy. Acta Biomater 61:114-123. ). Importantly, the combination of IL-12 with suicide gene therapy, such as HSVtk/GCV and CD/5’-FU seems to enhance the antitumor effect in pre-clinical and clinical studies in comparison to suicide gene therapy or IL-12 alone, increasing the presence of IL-12, IFNγ, in serum and tumor and inducing a specific antitumor immune response by NK cells and cytotoxic T cells activation in mouse model and in a phase I clinical trial (Freytag et al., 2013Freytag SO, Barton KN and Zhang Y (2013) Efficacy of oncolytic adenovirus expressing suicide genes and interleukin-12 in preclinical model of prostate cancer. Gene Ther 20:1131-1139. ; Barton et al., 2021Barton KN, Siddiqui F, Pompa R, Freytag SO, Khan G, Dobrosotskaya I, Ajlouni M, Zhang Y, Cheng J, Movsas B et al. (2021) Phase I trial of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for the treatment of metastatic pancreatic cancer. Mol Ther Oncolytics 20:94-104. ). Moreover, using IL-12 oncolytic adenovirus with selective replication in hypoxic conditions generated better antitumor response against pancreatic cancer in comparison to non-replicative adenovirus (Bortolanza et al., 2009Bortolanza S, Bunuales M, Otano I, Gonzalez-Aseguinolaza G, Ortiz-de-Solorzano C, Perez D, Prieto J and Hernandez-Alcoceba R (2009) Treatment of pancreatic cancer with an oncolytic adenovirus expressing interleukin-12 in Syrian hamsters. Mol Ther 17:614-622. ).

Differently from IL-24 and IL-12, IL-2 has already been approved by the Food and Drug Administration (FDA) for cancer treatment. It has also been demonstrated to be effective when delivered by an adenoviral vector in breast cancer using a specific promoter (Chaurasiya et al., 2016Chaurasiya S, Hew P, Crosley P, Sharon D, Potts K, Agopsowicz K, Long M, Shi C and Hitt MM (2016) Breast cancer gene therapy using an adenovirus encoding human IL-2 under control of mammaglobin promoter/enhancer sequences. Cancer Gene Ther 23:178-187. ). Other interleukins have also been tested in gene therapy against cancer, such as IL-15 oncolytic adenovirus in breast carcinoma (Yan Y et al., 2019Yan Z, McCray PB and Engelhardt JF (2019) Advances in gene therapy for cystic fibrosis lung disease. Hum Mol Genet 28:R88-R94. ), and AdIL-3 in prostate cancer in combination with radiotherapy (Oh et al., 2004Oh YT, Chen DWC, Dougherty GJ and McBride WH (2004) Adenoviral interleukin-3 gene-radiation therapy for prostate cancer in mouse model. Int J Radiat Oncol Biol Phys 59:579-583. ).

Several clinical trials evaluated the use of adenoviruses expressing cytokines for cancer treatment. A phase I clinical trial using Ad-IL12 for advanced digestive tumors demonstrated low toxicity, but only 29% of the patients presented disease stabilization and partial remission of the tumor in one patient. In addition, tumor immune infiltrate (CD4+ and CD8+ T cells) was observed in four of ten patients (Sangro et al., 2004Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I, Benito A, Larrache J, Pueyo J, Subtil JC et al. (2004) Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors. J Clin Oncol 22:1389-1397.). In advanced cancer patients, Ad-IL-24 was able to induced apoptosis in all tumors (Tong et al., 2005Tong AW, Nemunaitis J, Su D, Zhang Y, Cunningham C, Senzer N, Netto G, Rich D, Mhashilkar A, Parker K et al. (2005) Intratumoral injection of INGN241, a nonreplicative adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/IL24) biologic outcome in advanced cancer patients. Mol Ther 11:160-72.).

Using IL-2, adenoviral gene therapy in prostate cancer patiets was well tolerated; inducing tumor lymphocytic infiltration, increase in IFNγ and IL-4 secretion within the tumor, and decrease in prostate specific antigen (PSA) levels (Trudel et al., 2003Trudel S, Trachtenberg J, Toi A, Sweet L, Li ZH, Jewett M, Tchilias J, Zhuang LH, Hitt M, Wan Y et al. (2003) A phase I trial of adenovector-mediated delivery of interleukin-2 (AdIL-2) in high-risk localized prostate câncer. Cancer Gene Ther 10:755-763.). In melanoma and other solid tumors patients, Ad-IL-12 also induced tumor lymphocytic infiltration (Dummer et al., 2008Dummer R, Rochlitx C, Velu T, Acres B, Limacher JM, Bleuzen P, Lacoste G, Slos P, Romero P, Urosevic M (2008) Itralesional adenovirus-mediated interleukin-2 gene transfer for advanced solid cancers and melanoma. Mol Ther 16:985-94.). Another phase I study demonstrated safety, no severe adverse side effects and no presence of systemic IL-2. However, only 24% of the metastatic breast cancer and melanoma patients resulted in tumor regression and tumor lymphocytic infiltration (Stewart et al., 1999Stewart AK, Lassam NJ, Quirt IC, Bailey DJ, Rotstein LE, Krajden M, Dessureault D, Galinger S, Cappe D, Wan Y et al. (1999) Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial. Gene Ther 6:350-363. ). Additionally, an oncolytic adenovirus expressing TNFα and IL-2 is being currently (2022) tested in phase I clinical trials (See in clinicaltrials.govClinicalTrials.gov, gov, https://clinicaltrials.gov/ (accessed 21 June 2022).
https://clinicaltrials.gov/... ).

Different approaches for inducing an immune response against tumor cells using adenoviral vectors include the expression of CD40 ligand to promote the activation of adaptive immune response (Hanyu et al., 2008Hanyu K, Iida T, Shiba H, Ohashi T, Eto Y and Yanaga K (2008) Immunogene therapy by adenovirus vector expressing CD40 ligand for metastatic liver cancer in rats. Anticancer Res 28:2785-2789.; Vardouli et al., 2009Vardouli L, Lindqvist C, Vlahou K, Loskog ASI and Eliopoulos AG (2009) Adenovirus delivery of human CD40 ligand gene confers direct therapeutic effects on carcinomas. Cancer Gene Ther 16:848-860. ; Iida et al., 2010Iida T, Shiba H, Misawa T, Ohashi T, Eto Y and Yanaga K (2010) Immunogene therapy against colon cancer metastasis using an adenovirus vector expressing CD40 ligand. Surgery 148:925-935. ); NF-κB inhibition through the expression of its inhibitor, IκBα (Sumitomo et al., 1999Sumitomo M, Tachibana M, Ozu C, Asakura H, Murai M, Hayakawa M, Nakamura H, Takayanagi A and Shimizu N (1999) Induction of Apoptosis of Cytokine-Producing Bladder Cancer Cells by Adenovirus-Mediated I k B a Overexpression. Hum Gene Ther 10:37-47; Mukogawa et al., 2003Mukogawa T, Koyama F, Tachibana M, Takayanagi A, Shimizu N, Fujii H, Ueno M, Matsumoto H, Takeuchi T and Nakajima Y (2003) Adenovirus-mediated gene transduction of truncated IκBα α α α enhances radiosensitivity in human colon cancer cells. Cancer Sci 94:745-750.); and the expression of pathogen-associated molecular patterns (PAMPs) to trigger innate immune responses (Tosch et al., 2009Tosch C, Geist M, Ledoux C, Ziller-Remi C, Paul S, Erbs P, Corvaia N, Von Hoegen P, Balloul JM and Haegel H (2009) Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy. Cancer Gene Ther 16:310-319.). Oncolytic adenovirus expressing immunomodulatory genes like GM-CSF has the potential to destroy tumor cells and at the same time modulate the immune system in the tumor microenvironment, having been evaluated in clinical trials (Ranki et al., 2016Ranki T, Pesonen S, Hemminki A, Partanen K, Kairemo K, Alanko T, Lundin J, Linder N, Turkki R, Ristimäki A et al. (2016) Phase I study with ONCOS-102 for the treatment of solid tumors - an evaluation of clinical response and exploratory analyses of immune markers. J Immunother Cancer 4:17. ).

Therapeutic targets that involve the activation of the immune response within the tumor microenvironment may contribute to the activation of important pathways that lead to immune cell death and amplification of the destruction of the tumor. The use of adenovirus as gene carriers solves the problem of systemic contamination that leads to non-desired immune effects. Adenoviruses can increase the cytokine levels within the tumor, and decrease the systemic circulation. However, the use of non-replicative adenovirus expressing cytokines alone was not always successfull, but the combination with oncolytic adenovirus, radio-, chemotherapy or other therapies potentialized the antitumor effect.

Use of adenoviral vectors as vaccines

Replicative adenovirus was firstly used in the 1960s as a vaccine against respiratory disease in an enteric-coated tablet to elicit immune response in the intestinal tract, this way avoiding respiratory symptoms (Couch et al., 1963Couch RB, Chanock RM, Cate TR, Lang DJ, Kinght V and Huebner RJ (1963) Immunization with types 4 and 7 adenovirus by selective infection of the intestinal tract. Am Rev Respir Dis 88:394-403. ). Since these vaccines showed to be safe in humans, recombinant adenovirus started to be considered as a possible tool for vaccine development against other viral infections, like Hepatitis B virus and Human Immunodeficiency virus (HIV) (Morin et al., 1987Morin JE, Lubeck MD, Barton JE, Conley AJ, Davis AR and Hung PP (1987) Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters. Proc Natl Acad Sci U S A 84:4626-4630. ).

Adenovirus against HIV

The first articles proposing adenoviral vectors for HIV were published in the 1990s and parallel studies on Simian Immunodeficiency virus (SIV) were performed, however, in this review, we are focusing only on HIV results.

Initially, replicative Ad4, Ad5, and Ad7 were used to carry the sequence of HIV-1 envelope glycoprotein gene (env), or gag-protease gene (Chanda et al., 1990Chanda PK, Natuk RJ, Dheer SK, Lubeck MD, Bhat BM, Mason BB, Greenberg L, Mizutani S, Davis AR and Hung PP (1990) Helper independent recombinant adenovirus vectors: Expression of HIV env or HBV surface antigen. Int Rev Immunol 7:67-77. ; Natuk et al., 1992Natuk RJ, Chanda PK, Lubeck MD, Davis AR, Wilhelm J, Hjorth R, Wade MS, Bhat BM, Mizutani S, Lee S et al. (1992) Adenovirus-human immunodeficiency virus (HIV) envelope recombinant vaccines elicit high-titered HIV-neutralizing antibodies in the dog model. Proc Natl Acad Sci U S A 89:7777. ; Natuk et al., 1993Natuk RJ, Lubeck MD, Chanda PK, Chengalvala M, Wade MS, Murthy SCS, Wilhelm J, Vernon SK, Dheer SK, Mizutani S et al. (1993) Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees. AIDS Res Hum Retroviruses 9:395-404. ). These vectors were evaluated in dog, rhesus monkey and chimpanzee models and were capable of eliciting neutralizing antibodies against HIV (Natuk et al., 1992Natuk RJ, Chanda PK, Lubeck MD, Davis AR, Wilhelm J, Hjorth R, Wade MS, Bhat BM, Mizutani S, Lee S et al. (1992) Adenovirus-human immunodeficiency virus (HIV) envelope recombinant vaccines elicit high-titered HIV-neutralizing antibodies in the dog model. Proc Natl Acad Sci U S A 89:7777. ,1993Natuk RJ, Lubeck MD, Chanda PK, Chengalvala M, Wade MS, Murthy SCS, Wilhelm J, Vernon SK, Dheer SK, Mizutani S et al. (1993) Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees. AIDS Res Hum Retroviruses 9:395-404. ; Lubeck et al., 1994Lubeck MD, Natuk RJ, Chengalvala M, Chanda PK, Murthy KK, Murthy S, Mizutani S, Lee SG, Wade MS, Bhat BM et al. (1994) Immunogenicity of recombinant adenovirus-human immunodeficiency virus vaccines in chimpanzees following intranasal administration. AIDS Res Hum Retroviruses 10:1443-1449. ; Casimiro et al., 2003aCasimiro DR, Chen L, Fu T-M, Evans RK, Caulfield MJ, Davies M-E, Tang A, Chen M, Huang L, Harris V et al. (2003a) Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol 77:6305-6313. ). Alternatively, using the same vectors with HIV gp160 sequence, chimpanzees were protected against virus challenge (Lubeck et al., 1997Lubeck MD, Natuk R, Myagkikh M, Kalyan N, Aldrich K, Sinangil F, Alipanah S, Murthy SCS, Chanda PK, Nigida SM et al. (1997) Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization. Nat Med 3:651-658. ). Interestingly, cellular response was also achieved using non-replicative Ad5 to deliver HIV1 gag gene, a safer vaccine model (Casimiro et al., 2003bCasimiro DR, Tang A, Chen L, Fu T-M, Evans RK, Davies M-E, Freed DC, Hurni W, Aste-Amezaga JM, Guan L et al. (2003b) Vaccine-induced immunity in baboons by using DNA and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol 77:7663-7668. ). After this, replicative-defective adenoviruses were extensively used and the results discussed next are from these first-generation vectors.

Using a vaccination protocol of DNA vaccine prime and Ad5 or Ad5/35 expressing env gene as a boost in mice, the authors observed it induced high levels of IFNγ-secreting cells (Takakura et al., 2005Takakura M, Okuda K, Matsuda T, Takeshita F, Takakura H, Ikezawa Z and Xin KQ (2005) Combination of DNA vaccine and adenovirus vector by cutaneous administration induced strong HIV-specific cellular immune responses in mice. Vaccine 23:847-848. ), neutralizing antibodies (Mascola et al., 2005Mascola JR, Sambor A, Beaudry K, Santra S, Welcher B, Louder MK, Vancott TC, Huang Y, Chakrabarti BK, Kong W-P et al. (2005) Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficiency virus type 1 proteins. J Virol 79:771-779. ) and protection against a recombinant HIV-vaccinia virus (Xin et al., 2005Xin KQ, Jounai N, Someya K, Honma K, Mizuguchi H, Naganawa S, Kitamura K, Hayakawa T, Saha S, Takeshita F et al. (2005) Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV. Gene Ther 12:1769-1777. ). Similar results were obtained using other heterologous systems, such as Ad5/poxvirus vectors with HIV gag in rhesus macaques (Casimiro et al., 2004Casimiro DR, Bett AJ, Fu T, Davies M-E, Tang A, Wilson KA, Chen M, Long R, McKelvey T, Chastain M et al. (2004) Heterologous Human Immunodeficiency Virus Type 1 Priming-Boosting Immunization Strategies Involving Replication-Defective Adenovirus and Poxvirus Vaccine Vectors. J Virol 78:11434); Ad5 or Ad7 followed by HIV gp120 protein immunization in chimpanzees (Gómez-Román et al., 2006Gómez-Román VR, Florese RH, Peng B, Montefiori DC, Kalyanaraman VS, Venzon D, Srivastava I, Barnett SW and Robert-Guroff M (2006) An adenovirus-based HIV subtype B prime/boost vaccine regimen elicits antibodies mediating broad antibody-dependent cellular cytotoxicity against non-subtype B HIV strains. J Acquir Immune Defic Syndr 43:270-277. ); DNA/Ad5/protein in rhesus macaques (Vinner et al., 2006Vinner L, Therrien D, Wee E, Laursen I, Hanke T, Corbet SL and Fomsgaard A (2006) Immune response in rhesus macaques after mixed modality immunisations with DNA, recombinant adenovirus and recombinant gp120 from human immunodeficiency virus type 1. APMIS 114:690-699. ) or guinea pigs (Shu et al., 2007Shu Y, Winfrey S, Yang Z-Y, Xu L, Rao SS, Srivastava I, Barnett SW, Nabel GJ and Mascola JR (2007) Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs. Vaccine 25:1398. ); DNA/Ad5/Sendai virus carrying HIV gag tested in mice and rhesus macaques (Yu et al., 2008Yu S, Feng X, Shu T, Matano T, Hasegawa M, Wang X, Ma H, Li H, Li Z and Zeng Y (2008) Potent specific immune responses induced by prime-boost-boost strategies based on DNA, adenovirus, and Sendai virus vectors expressing gag gene of Chinese HIV-1 subtype B. Vaccine 26:6124-6131. ) and lentivirus/Ad5 in mice (Asefa et al., 2010Asefa B, Korokhov N and Lemiale F (2010) Heterologous HIV-based lentiviral/adenoviral vectors immunizations result in enhanced HIV-specific immunity. Vaccine 28:3617-3624. ).

After tests in several animal models, the first phase I clinical trials started to show results, healthy adults were inoculated with a mixture of 4 recombinant Ad5 for 3 different clades of HIV1 (Catanzaro et al., 2006Catanzaro AT, Koup RA, Roederer M, Bailer RT, Enama ME, Moodie Z, Gu L, Martin JE, Novik L, Chakrabarti BK et al. (2006) Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector. J Infect Dis 194:1638-1649) or Ad5 with HIV-1 Clade B gag/pol/nef (Priddy et al., 2008Priddy FH, Brown D, Kublin J, Monahan K, Wright DP, Lalezari J, Santiago S, Marmor M, Lally M, Novak RM et al. (2008) Safety and immunogenicity of a replication-incompetent adenovirus type 5 HIV-1 clade B gag/pol/nef vaccine in healthy adults. Clin Infect Dis 46:1769-1781. ) and there were no major concerns about safety. Around 2007 some clinical trials resulted in no protection against HIV, while other clinical trials indicated possible problems involving previous Ad5 infection, emerging evidence showed that previous exposure to adenovirus could impair vaccine efficacy (Steinbrook, 2007Steinbrook R (2007) One step forward, two steps back--will there ever be an AIDS vaccine? N Eng J Med 357:2653-2655. ; Quirk et al., 2008Quirk EK, Mogg R, Brown DD, Lally MA, Mehrotra DV, DiNubile MJ and Robertson MN (2008) HIV seroconversion without infection after receipt of adenovirus-vectored HIV type 1 vaccine. Clin Infect Dis 47:1593-1599. ; Sekaly, 2008Sekaly RP (2008) The failed HIV Merck vaccine study: a step back or a launching point for future vaccine development? J Exp Med 205:7. ; Yu et al., 2008Yu S, Feng X, Shu T, Matano T, Hasegawa M, Wang X, Ma H, Li H, Li Z and Zeng Y (2008) Potent specific immune responses induced by prime-boost-boost strategies based on DNA, adenovirus, and Sendai virus vectors expressing gag gene of Chinese HIV-1 subtype B. Vaccine 26:6124-6131. ). Even worse, Ad5 seropositive individuals vaccinated could have a more permissive environment for HIV infection (Perreau et al., 2008Perreau M, Pantaleo G and Kremer EJ (2008) Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells. J Exp Med 205:2717. ; Benlahrech et al., 2009Benlahrech A, Harris J, Meiser A, Papagatsias T, Hornig J, Hayes P, Lieber A, Athanasopoulos T, Bachy V, Csomor E et al. (2009) Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1. Proc Natl Acad Sci U S A 106:19940-19945. ; D’Souza and Frahm, 2010D´Souza MP and Frahm N (2010) Adenovirus 5 serotype vector-specific immunity and HIV-1 infection: a tale of T cells and antibodies. AIDS 24:803-809; Hu et al., 2014Hu H, Eller MA, Zafar S, Zhou Y, Gu M, Wei Z, Currier JR, Marovich MA, Kibuuka HN, Bailer RT et al. (2014) Preferential infection of human Ad5-specific CD4 T cells by HIV in Ad5 naturally exposed and recombinant Ad5-HIV vaccinated individuals. Proc Natl Acad Sci U S A 111:13439-13444. ), despite some controversial results (O’Brien et al., 2009O’Brien KL, Liu J, King SL, Sun YH, Schmitz JE, Lifton MA, Hutnick NA, Betts MR, Dubey SA, Goudsmit J et al. (2009) Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans. Nat Med 15:873-875. ; Curlin et al., 2011Curlin ME, Cassis-Ghavami F, Magaret AS, Spies GA, Duerr A, Celum CL, Sanchez JL, Margolick JB, Detels R, McElrath MJ et al. (2011) Serological Immunity to Adenovirus serotype 5 is not Associated with Risk of HIV Infection: a Case-control Study. AIDS (London) 25:153. ; Kaner et al., 2012Kaner RJ, Santiago F, Rahaghi F, Michaels E, Moore JP and Crystal RG (2012) Adenovirus Vectors Block Human Immunodeficiency Virus-1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat. Am J Respir Cell Mol Biol 43:234-242.). In spite of the increased susceptibility to HIV infection, later analysis showed no difference in disease progression between Ad5 vaccinated and placebo groups (Fitzgerald et al., 2011Fitzgerald DW, Janes H, Robertson M, Coombs R, Frank I, Gilbert P, Loufty M, Mehrotra D and Duerr A (2011) An Ad5-Vectored HIV-1 Vaccine Elicits Cell-mediated Immunity but does not Affect Disease Progression in HIV-1-infected Male Subjects: Results From a Randomized Placebo-Controlled Trial (The Step Study). J Infect Dis 203:765. ). One explanation for such increased susceptibility for HIV infection in the Ad5 vaccinated individuals is that stimulus with Ad5 in preexistent Ad5-seropositive individuals may trigger expansion of a specific HIV susceptible CD4+ population with increased CCR5 expression, the co-receptor used by HIV to infect the cells (Benlahrech et al., 2009) and that have a Th17-like phenotype (Hu et al., 2014Hu H, Eller MA, Zafar S, Zhou Y, Gu M, Wei Z, Currier JR, Marovich MA, Kibuuka HN, Bailer RT et al. (2014) Preferential infection of human Ad5-specific CD4 T cells by HIV in Ad5 naturally exposed and recombinant Ad5-HIV vaccinated individuals. Proc Natl Acad Sci U S A 111:13439-13444. ).

Because of the negative results, alternatives were investigated and other types of adenoviruses in animal models started to be employed (Michael 2012Michael NL (2012) Rare serotype adenoviral vectors for HIV vaccine development. J Clin Invest 122:25-27. ), for example using chimpanzee adenovirus (ChAd) (Santra et al., 2009Santra S, Sun Y, Korioth-Schmitz B, Fitzgerald J, Charbonneau C, Santos G, Seaman MS, Ratcliffe SJ, Montefiori DC, Nabel GJ et al. (2009) Heterologous Prime/Boost Immunizations of Rhesus Monkeys Using Chimpanzee Adenovirus Vectors. Vaccine 27:5837. ); ovine adenovirus (OAd) (Bridgeman et al., 2009Bridgeman A, Roshorm Y, Lockett LJ, Xu ZZ, Hopkins R, Shaw J, Both GW and Hanke T (2009) Ovine atadenovirus, a novel and highly immunogenic vector in prime-boost studies of a candidate HIV-1 vaccine. Vaccine 28:474-483. ); Ad26 expressing HIV-1 Gag, Pol, and Env antigens (Barouch et al., 2010Barouch DH, O’Brien KL, Simmons NL, King SL, Abbink P, Maxfield LF, Sun YH, la Porte A, Riggs AM, Lynch DM et al. (2010) Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat Med 16:319-323. ); Ad4-Env (Alexander et al., 2013Alexander J, Mendy J, Vang L, Avanzini JB, Garduno F, Manayani DJ, Ishioka G, Farness P, Ping LH, Swanstrom R et al. (2013) Pre-clinical development of a recombinant, replication-competent adenovirus serotype 4 vector vaccine expressing HIV-1 envelope 1086 clade C. PloS One 8:e82380. ) or even edited/mutated Ad5 (Gabitzsch et al., 2009Gabitzsch ES, Xu Y, Yoshida LH, Balint J, Amalfitano A and Jones FR (2009) Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag, Pol, Nef despite the presence of Ad5 immunity. Vaccine 27:6394-6398. ; Hidajat et al., 2010Hidajat R, Kuate S, Venzon D, Kalyanaraman V, Kalisz I, Treece J, Lian Y, Barnett SW and Robert-Guroff M(2010) Construction and immunogenicity of replication-competent adenovirus 5 host range mutant recombinants expressing HIV-1 gp160 of SF162 and TV1 strains. Vaccine28:3963-3971. ). Other prime-boost regimens were developed, like combinations of Bacillus Calmette-Guérin (BCG)/OAd/poxvirus (Hopkins et al., 2011Hopkins R, Bridgeman A, Bourne C, Mbewe-Mvula A, Sadoff JC, Both GW, Joseph J, Fulkerson J and Hanke T (2011) Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors. Eur J Immunol 41:3542-3552. ); Ad26/Ad35 (Barouch et al., 2012Barouch DH, Klasse PJ, Dufour J, Veazey RS and Moore JP (2012) Macaque studies of vaccine and microbicide combinations for preventing HIV-1 sexual transmission. Proc Natl Acad Sci U S A 109:8694-8698. ; Kaufman et al., 2012Kaufman DR, Li F, Cruz AN, Self SG and Barouch DH (2012) Focus and Breadth of Cellular Immune Responses Elicited by a Heterologous Insert Prime-Boost Vaccine Regimen in Rhesus Monkeys. Vaccine 30:506. ); ChAd/DNA/modified Vaccinia virus Ankara (MVA) (Roshorm et al., 2012Roshorm Y, Cottingham MG, Potash MJ, Volsky DJ and Hanke T (2012) T cells induced by recombinant chimpanzee adenovirus alone and in prime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load. Eur J Immunol 42:3243-3255. ); Ad35/MVA (Ratto-Kim et al., 2012Ratto-Kim S, Currier JR, Cox JH, Excler JL, Valencia-Micolta A, Thelian D, Lo V, Sayeed E, Polonis VR, Earl PL et al. (2012) Heterologous Prime-Boost Regimens Using rAd35 and rMVA Vectors Elicit Stronger Cellular Immune Responses to HIV Proteins Than Homologous Regimens. PloS One 7:e45840. ). Additionally, second-generation adenoviruses were also used, based on Ad5 (Thomas et al., 2013Thomas MA, Song R, Demberg T, Vargas-Inchaustegui DA, Venzon D and Robert-Guroff M (2013) Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors. PloS One 8:e76344). The delivery route of vaccination could be an alternative as well, as respiratory aerosolization delivery (Kaufman et al., 2010Kaufman DR, Bivas-Benita M, Simmons NL, Miller D and Barouch DH (2010) Route of Adenovirus-Based HIV-1 Vaccine Delivery Impacts the Phenotype and Trafficking of Vaccine-Elicited CD8 + T Lymphocytes. J Virol 84:5986-5996. ) or sublingual vaccination appeared to enhance CD8⁺ T cells activation, especially in mucosal sites (Appledorn et al., 2011Appledorn DM, Aldhamen YA, Godbehere S, Seregin SS and Amalfitano A (2011) Sublingual administration of an adenovirus serotype 5 (Ad5)-based vaccine confirms Toll-like receptor agonist activity in the oral cavity and elicits improved mucosal and systemic cell-mediated responses against HIV antigens despite preexisting Ad5 immunity. Clin Vaccine Immunol 18:150-160. ).

Clinical trials in healthy adults were conducted for Ad35 containing multiple HIV genes (Keefer et al., 2012Keefer MC, Gilmour J, Hayes P, Gill D, Kopycinski J, Cheeseman H, Cashin-Cox M, Naarding M, Clark L, Fernandez N et al. (2012) A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults. PloS One7:e41936. ; Kopycinski et al., 2014Kopycinski J, Hayes P, Ashraf A, Cheeseman H, Lala F, Czyzewska-Khan J, Spentzou A, Gill DK, Keefer MC, Excler JL et al. (2014) Broad HIV epitope specificity and viral inhibition induced by multigenic HIV-1 Adenovirus Subtype 35 vector vaccine in healthy uninfected adults. PloS One 9:e90378. ; Omosa-Manyonyi et al., 2015Omosa-Manyonyi G, Mpendo J, Ruzagira E, Kilembe W, Chomba E, Roman F, Bourguignon P, Koutsoukos M, Collard A, Voss G et al. (2015) A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults. PloS One 10:e0125954. ); Ad26-Env (Baden et al., 2013Baden LR, Walsh SR, Seaman MS, Tucker RP, Krause KH, Patel A, Johnson JA, Kleinjan J, Yanosick KE, Perry J et al. (2013) First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis 207:240-247., 2015Baden LR, Liu J, Li H, Johnson JA, Walsh SR, Kleinjan JA, Engelson BA, Peter L, Abbink P, Milner DA et al. (2015) Induction of HIV-1-specific mucosal immune responses following intramuscular recombinant adenovirus serotype 26 HIV-1 vaccination of humans. J Infect Dis 211:518-528. ; Barouch et al., 2013Barouch DH, Liu J, Peter L, Abbink P, Iampietro MJ, Cheung A, Alter G, Chung A, Dugast AS, Frahm N et al. (2013) Characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype 26 HIV-1 Env vaccine in healthy adults (IPCAVD 001). J Infect Dis 207:248-256. ; Esparza, 2013Esparza J (2013) Progress in the development of an adenovirus 26 vector platform for HIV vaccines. Expert Rev Vaccines 12:477-480. ); Ad5 modified with Ad48 hexon expressing HIV env (Ad5HVR48-Env) (Baden et al., 2014Baden LR, Walsh SR, Seaman MS, Johnson JA, Tucker RP, Kleinjan JA, Gothing JA, Engelson BA, Carey BR, Oza A et al. (2014) First-in-human evaluation of a hexon chimeric adenovirus vector expressing HIV-1 Env (IPCAVD 002). J Infect Dis 210:1052-1061. ); a heterologous system with DNA, followed by ChAd and MVA, all carrying a fusion of all HIV conserved antigens (Hayton et al., 2014Hayton EJ, Rose A, Ibrahimsa U, Sorbo M del, Capone S, Crook A, Black AP, Dorrell L and Hanke T (2014) Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial. PloS One 9:e101591. ); a regimen of prime-boost with Ad35/Ad5 (Fuchs et al., 2015Fuchs JD, Bart PA, Frahm N, Morgan C, Gilbert PB, Kochar N, DeRosa SC, Tomaras GD, Wagner TM, Baden LR et al. (2015) Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals. J AIDS Clin Res6:461. ; Crank et al., 2016Crank MC, Wilson EMP, Novik L, Enama ME, Hendel CS, Gu W, Nason MC, Bailer RT, Nabel GJ, McDermott AB et al. (2016) Safety and Immunogenicity of a rAd35-EnvA Prototype HIV-1 Vaccine in Combination with rAd5-EnvA in Healthy Adults (VRC 012). PloS One 11:e0166393. ; Walsh et al., 2016bWalsh SR, Moodie Z, Fiore-Gartland AJ, Morgan C, Wilck MB, Hammer SM, Buchbinder SP, Kalams SA, Goepfert PA, Mulligan MJ et al. (2016b) Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083. J Infect Dis 213:541-550. ); Ad26/Ad35 (Baden et al., 2016Baden LR, Karita E, Mutua G, Bekker LG, Gray G, Page-Shipp L, Walsh SR, Nyombayire J, Anzala O, Roux S et al. (2016) Assessment of the Safety and Immunogenicity of 2 Novel Vaccine Platforms for HIV-1 Prevention: A Randomized Trial. Ann Intern Med 164:313-322. ) or Sendai virus/Ad35. In a phase 3 trial, DNA prime with Ad5 boost showed no efficacy in a high risk for HIV1 infection population (Nyombayire et al., 2017Nyombayire J, Anzala O, Gazzard B, Karita E, Bergin P, Hayes P, Kopycinski J, Omosa-Manyonyi G, Jackson A, Bizimana J et al. (2017) First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus-Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens. J Infect Dis 215:95-104. ), however high levels of specific CD8⁺ T cells were described to be associated with a lower risk of HIV infection (Janes et al., 2017Janes HE, Cohen KW, Frahm N, de Rosa SC, Sanchez B, Hural J, Magaret CA, Karuna S, Bentley C, Gottardo R et al. (2017) Higher T-Cell Responses Induced by DNA/rAd5 HIV-1 Preventive Vaccine Are Associated With Lower HIV-1 Infection Risk in an Efficacy Trial. Journal Infect Dis 215:1376-1385. ). Recently, an Ad26 expressing Env/Gag/Pol in a 2b Clinical trial failed to confer high protection against HIV, showing about 25% of vaccine efficacy (CISION PR Newswire, 2022CISION PR Newswire, CISION PR Newswire, https://www.prnewswire.com/news-releases/johnson--johnson-and-global-partners-announce-results-from-phase-2b-imbokodo-hiv-vaccine-clinical-trial-in-young-women-in-sub-saharan-africa-301365918.html (accessed 12 September 2022).
https://www.prnewswire.com/news-releases... ). Several other studies with other prime-boost formulations are underway and may provide better results.

Adenovirus against coronavirus

Another beneficiary of adenoviral vectors development is the vaccine against coronavirus. The first adenoviral vector that provided protection against a coronavirus was tested in pigs in 1994. The non-replicative Ad5 was used to carry the glycoprotein S (Spike) of porcine respiratory coronavirus (PRCV) and elicited mucosal immunity in pigs (Callebaut et al., 1993Callebaut P, Pensaert M and Enjuanes L (1993) Construction of a recombinant adenovirus for the expression of the glycoprotein S antigen of porcine respiratory coronavirus. Adv Exp Med Biol 342:469-470. ). The animals produced neutralizing antibodies against PRCV (Callebaut and Pensaert, 1995Callebaut P and Pensaert M (1995) Expression and immunogenicity of the spike glycoprotein of porcine respiratory coronavirus encoded in the E3 region of adenovirus. Adv Exp Med Biol 380:265-270. ; Callebaut et al., 1996Callebaut P, Enjuanes L and Pensaert M (1996) An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine. J Gen Virol 77:309-313. ). Another adenoviral vector was constructed carrying haemagglutinin-esterase (HE) of bovine coronavirus (BCV) and tested in cotton rats; it induced systemic and mucosal immune responses after immunization (Baca-Estrada et al., 1995Baca-Estrada ME, Liang X, Babiuk LA and Yoo & D (1995) Induction of mucosal immunity in cotton rats to haemagglutinin-esterase glycoprotein of bovine coronavirus by recombinant adenovirus. Immunology 86:134.). In the following years, other adenoviral vectors expressing coronavirus proteins from different animal species were tested including transmissible gastroenteritis coronavirus (TGEV) (Torres et al., 1995Torres JM, Sánchez C, Suñé C, Smerdou C, Prevec L, Graham F and Enjuanes L (1995) Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein. Virology 213:503-516.; Torres et al., 1996Torres JM, Alonso C, Ortega A, Mittal S, Graham F and Enjuanes L (1996) Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus. J Virol 70:3770-3780. ).

The outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV-1) in 2003 stimulated researchers to find effective vaccines against coronavirus infecting humans. Pre-clinical studies in mice and rats showed potent immune responses against the nucleocapsid (N) and Spike (S) proteins of SARS-CoV-1 after their delivery by an Ad5 vector (Liu et al., 2005Liu RY, Wu LZ, Huang BJ, Huang JL, Zhang YL, Ke M la, Wang JM, Tan WP, Zhang RH, Chen HK et al. (2005) Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats. Virus Res 112:24-31. ; Zakhartchouk et al., 2005Zakhartchouk AN, Viswanathan S, Mahony JB, Glaudei J and Babiuk LA (2005) Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice. J Gen Virol 86:211-215. ; See et al., 2006See RH, Zakhartchouk AN, Petric M, Lawrence DJ, Mok CPY, Hogan RJ, Rowe T, Zitzow LA, Karunakaran KP, Hitt MM et al. (2006) Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus. J Gen Virol 87:641-650. ; Shim et al., 2012Shim BS, Stadler K, Nguyen HH, Yun CH, Kim DW, Chang J, Czerkinsky C and Song MK (2012) Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain. Virol J 9:215. ). Chimpanzee adenovirus C7 (ChAdC7) vector was also tested and elicited immune response against SARS-CoV1 in mice (Zhi et al., 2006Zhi Y, Figueredo J, Kobinger GP, Hagan H, Calcedo R, Miller JR, Gao G and Wilson JM (2006) Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus. Hum Gene Ther 17:500-506. ).

In 2012, a new outbreak was caused by the Middle East respiratory syndrome coronavirus (MERS-CoV). Since then, there have been several studies showing neutralizing antibodies produced in mice immunized with human Ad5, Ad26, Ad41 or ChAd carrying the S gene (Kim et al., 2014Kim E, Okada K, Kenniston T, Raj VS, AlHajri MM, Farag EABA, AlHajri F, Osterhaus ADME, Haagmans BL and Gambotto A (2014) Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice. Vaccine32:5975-5982. ; Guo et al., 2015Guo X, Deng Y, Chen H, Lan J, Wang W, Zou X, Hung T, Lu Z and Tan W (2015) Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus. Immunology145:476-484. ; Alharbi et al., 2017Alharbi NK, Padron-Regalado E, Thompson CP, Kupke A, Wells D, Sloan MA, Grehan K, Temperton N, Lambe T, Warimwe G et al. (2017) ChAdOx1 and MVA based vaccine candidates against MERS-CoV elicit neutralising antibodies and cellular immune responses in mice. Vaccine 35:3780-3788; Jung et al., 2018Jung SY, Kang KW, Lee EY, Seo DW, Kim HL, Kim H, Kwon TW, Park HL, Kim H, Lee SM et al. (2018) Heterologous prime-boost vaccination with adenoviral vector and protein nanoparticles induces both Th1 and Th2 responses against Middle East respiratory syndrome coronavirus. Vaccine36:3468-3476. ; Jia et al., 2019Jia W, Channappanavar R, Zhang C, Li M, Zhou H, Zhang S, Zhou P, Xu J, Shan S, Shi X et al. (2019) Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection. Emerg Microbes Infect 8:760-772. ; Dolzhikova et al., 2020Dolzhikova IV, Grousova DM, Zubkova OV, Tukhvatulin AI, Kovyrshina AV, Lubenets NL, Ozharovskaia TA, Popova O, Esmagambetov IB, Shcheblyakov DV et al. (2020) Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome. Acta naturae 12:114-123. ). ChAd-S was later tested in camels, the natural host of MERS-CoV, and induced production of neutralizing antibodies (Alharbi et al., 2019Alharbi NK, Qasim I, Almasoud A, Aljami HA, Alenazi MW, Alhafufi A, Aldibasi OS, Hashem AM, Kasem S, Albrahim R et al. (2019) Humoral Immunogenicity and Efficacy of a Single Dose of ChAdOx1 MERS Vaccine Candidate in Dromedary Camels. Sci Rep 9:16292. ).

ChAd vectors, including ChAdOX1 developed by researchers from Oxford University, were then redirected to be used against the new pandemic coronavirus, SARS-CoV-2 in 2020. In rhesus macaques, mice, hamster and ferret’ models, the ChAd vectors carrying the S gene were able to induce robust immune responses and protect animals from pneumonia, results that greatly contributed to fast-track vaccines to the first clinical trials (van Doremalen et al., 2020van Doremalen N, Lambe T, Spencer A, Belij-Rammerstorfer S, Purushotham JN, Port JR, Avanzato VA, Bushmaker T, Flaxman A, Ulaszewska M et al. (2020) ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature 586:578-582. ; Hassan et al., 2020Hassan AO, Kafai NM, Dmitriev IP, Fox JM, Smith BK, Harvey IB, Chen RE, Winkler ES, Wessel AW, Case JB et al. (2020) A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2. Cell 183:169-184.e13.; 2021aHassan AO, Feldmann F, Zhao H, Curiel DT , Okumura A, Tang-Huau TL, Case JB, Meade-White K, Callison J, Chen RE et al. (2021a) A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques. Cell Rep Med 2:100230.,bHassan AO, Shrihari S, Gorman MJ, Ying B, Yaun D, Raju S, Chen RE, Dmitriev IP, Kashentseva E, Adams LJ et al. (2021b) An intranasal vaccine durably protects against SARS-CoV-2 variants in mice. Cell Rep 36:109452; Marsh et al., 2021Marsh GA, McAuley AJ, Au GG, Riddell S, Layton D, Singanallur NB, Layton R, Payne J, Durr PA, Bender H et al. (2021) ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets. NPJ Vaccines 6:1-8. ; Bricker et al., 2021Bricker TL, Darling TL, Hassan AO, Harastani HH, Soung A, Jiang X, Dai YN, Zhao H, Adams LJ, Holtzman MJ et al. (2021) A single intranasal or intramuscular immunization with chimpanzee adenovirus-vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters. Cell Rep 36:109400. ). The same effects were observed in rhesus macaques and hamsters using human Ad26 as a carrier for the S gene (Mercado et al., 2020Mercado NB, Zahn R, Wegmann F, Loos C, Chandrashekar A, Yu J, Liu J, Peter L, McMahan K, Tostanoski LH et al. (2020) Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature 586:583-588. ; Tostanoski et al., 2020Tostanoski LH, Wegmann F, Martinot AJ, Loos C, McMahan K, Mercado NB, Yu J, Chan CN, Bondoc S, Starke CE et al. (2020) Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters. Nat Med 26:1694-1700. ; He et al., 2021He X, Chandrashekar A, Zahn R, Wegmann F, Yu J, Mercado NB, McMahan K, Martinot AJ, Piedra-Mora C, Beecy S et al. (2021) Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques. Cell184: 3467-3473.e11. ), and in mice and macaques using Ad5 (Wu et al., 2020Wu S, Zhong G, Zhang J, Shuai L, Zhang Z, Wen Z, Wang B, Zhao Z, Song X, Chen Y et al. (2020) A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge. Nat Commun 2020 11:4081. ; Feng et al., 2020Feng L, Wang Q, Shan C, Yang C, Feng Y, Wu J, Liu X, Zhou Y, Jiang R, Hu P et al. (2020) An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques. Nat Commun 11:4207. ; Kim et al., 2021Kim E, Weisel FJ, Balmert SC, Khan MS, Huang S, Erdos G, Kenniston TW, Carey CD, Joachim SM, Conter LJ et al. (2021) A single subcutaneous or intranasal immunization with adenovirus-based SARS-CoV-2 vaccine induces robust humoral and cellular immune responses in mice. Eur J Immunol 51:1774-1784. ; King et al., 2021King RG, Silva-Sanchez A, Peel JN, Botta D, Dickson AM, Pinto AK, Meza-Perez S, Allie SR, Schultz MD, Liu M et al. (2021) Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge. Vaccines9:881. ). In parallel, other vectors have been tested, such as simian adenovirus types 23 and 49 (Luo et al., 2021Luo S, Zhang P, Liu B, Yang C, Liang C, Wang Q, Zhang L, Tang X, Li J, Hou S et al. (2021) Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates. Emerg Microbes Infect 10:1002-1015. ), gorilla adenovirus 32 (Capone et al., 2021Capone S, Raggioli A, Gentile M, Battella S, Lahm A, Sommella A, Contino AM, Urbanowicz RA, Scala R, Barra F et al. (2021) Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19. Mol Ther 29:2412-2423) and rhesus adenovirus type 52 (Tostanoski et al., 2021Tostanoski LH, Gralinski LE, Martinez DR, Schaefer A, Mahrokhian SH, Li Z, Nampanya F, Wan H, Yu J, Chang A et al. (2021) Protective Efficacy of Rhesus Adenovirus COVID-19 Vaccines against Mouse-Adapted SARS-CoV-2. J Virol 95:e0097421. ).

Clinical trials were done in healthy adult volunteers using ChAd against MERS-CoV (Folegatti et al., 2020aFolegatti PM, Bittaye M, Flaxman A, Lopez FR, Bellamy D, Kupke A, Mair C, Makinson R, Sheridan J, Rohde C et al. (2020a) Safety and immunogenicity of a candidate Middle East respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial. Lancet Infect Dis 20:816-826. ). In order to protect from SARS-CoV-2 and prevent development of COVID19 disease, Ad5 (Zhu et al., 2020Zhu FC, Guan XH, Li YH, Huang JY, Jiang T, Hou LH, Li JX, Yang BF, Wang L, Wang WJ et al. (2020) Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 396:479-488.; Guzmán-Martínez et al., 2021Guzmán-Martínez O, Guardado K, de Guevara EL, Navarro S, Hernández C, Zenteno-Cuevas R and Montero H (2021) IgG Antibodies Generation and Side Effects Caused by Ad5-nCoV Vaccine (CanSino Biologics) and BNT162b2 Vaccine (Pfizer/BioNTech) among Mexican Population. Vaccines (Basel) 9:999. ; Wu et al., 2021Wu S, Huang J, Zhang Z, Wu J, Zhang J, Hu H, Zhu T, Zhang J, Luo L, Fan P et al. (2021) Safety, tolerability, and immunogenicity of an aerosolised adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in adults: preliminary report of an open-label and randomised phase 1 clinical trial. Lancet Infect Dis 21:1654-1664. ) (CT1, CT8, CT12), ChAdOx-1 (Folegatti et al., 2020bFolegatti PM, Ewer KJ, Aley PK, Angus B, Becker S, Belij-Rammerstorfer S, Bellamy D, Bibi S, Bittaye M, Clutterbuck EA et al. (2020b) Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet 396:467-478. ; Ramasamy et al., 2020Ramasamy MN, Minassian AM, Ewer KJ, Flaxman AL, Folegatti PM, Owens DR, Voysey M, Aley PK, Angus B, Babbage G et al. (2020) Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): A single-blind, randomised, controlled, phase 2/3 trial. Lancet 396:1979-1993. ), Ad26 (Tukhvatulin et al., 2021Tukhvatulin AI, Dolzhikova IV, Shcheblyakov DV, Zubkova OV, Dzharullaeva AS, Kovyrshina AV, Lubenets NL, Grousova DM, Erokhova AS, Botikov AG et al. (2021) An open, non-randomised, phase 1/2 trial on the safety, tolerability, and immunogenicity of single-dose vaccine “Sputnik Light” for prevention of coronavirus infection in healthy adults. Lancet Reg Health Eur 11:100241. ), or Ad26 and Ad5 as a prime-boost system (Logunov et al., 2020Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, Grousova DM, Erokhova AS, Kovyrshina AV, Botikov AG et al. (2020) Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet 396:887-897. ) have been employed; also ChAd was tested in health care workers (Benning et al., 2021Benning L, Töllner M, Hidmark A, Schaier M, Nusshag C, Kälble F, Reichel P, Buylaert M, Grenz J, Ponath G et al. (2021) Heterologous ChAdOx1 nCoV-19/BNT162b2 Prime-Boost Vaccination Induces Strong Humoral Responses among Health Care Workers. Vaccines (Basel) 9:857. ; Havervall et al., 2021Havervall S, Marking U, Greilert-Norin N, Ng H, Gordon M, Salomonsson AC, Hellström C, Pin E, Blom K, Mangsbo S et al. (2021) Antibody responses after a single dose of ChAdOx1 nCoV-19 vaccine in healthcare workers previously infected with SARS-CoV-2. EBioMedicine 70:103523. ; Lee et al., 2021Lee SW, Moon JY, Lee SK, Lee H, Moon SH, Chung SJ, Yeo Y, Park TS, Park DW, Kim TH et al. (2021) Anti-SARS-CoV-2 Spike Protein RBD antibody levels after receiving a second dose of ChAdOx1 nCov-19 (AZD1222) vaccine in healthcare workers: lack of association with age, sex, obesity, and adverse Reactions. Front Immunol 12:779212. ); Ad5 was evaluated in children above 6 years old (Zhu et al., 2021Zhu F, Jin P, Zhu T, Wang W, Ye H, Pan H, Hou L, Li J, Wang X, Wu S et al. (2021) Safety and immunogenicity of a recombinant adenovirus type-5-vectored COVID-19 vaccine with a homologous prime-boost regimen in healthy participants aged 6 years and above: a randomised, double-blind, placebo-controlled, phase 2b trial. Clin Infect Dis 75:e783-e791. ); and ChAd in heart transplanted individuals (Tanner et al., 2022Tanner R, Starr N, Chan G, Dempsey E, Heffernan E, Newman E, O’Neill J, Hannan MM, Lynch B and Joyce E (2022) Humoral response to SARS-CoV-2 adenovirus vector vaccination (ChAdOx1 nCoV-19 [AZD1222]) in heart transplant recipients aged 18 to 70 years of age. J Heart Lung Transplant 41:492-500. ). Showing its safety and effectiveness.

In phase 3 clinical trials, the heterologous prime-boost system using Ad26 and Ad5 as vectors presented an efficacy of 91,6% against COVID-19 (Logunov et al., 2021Logunov DY, Dolzhikova IV, Shcheblyakov DV, Tukhvatulin AI, Zubkova OV, Dzharullaeva AS, Kovyrshina AV, Lubenets NL, Grousova DM, Erokhova AS et al. (2021) Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in Russia. Lancet 397:671-681. ); injection of Ad26 alone, showed efficacy of 81.7% against severe-critical COVID-19 after 28 days of immunization (Sadoff et al., 2021Sadoff J, Gray G, Vandebosch A, Cárdenas V, Shukarev G, Grinsztejn B, Goepfert PA, Truyers C, Fennema H, Spiessens B et al. (2021) Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19. N Engl J Med 384:2187-2201. ). Using one dose of Ad5 alone, efficacy against symptomatic infection was 57.5% (Halperin et al., 2022Halperin SA, Ye L, MacKinnon-Cameron D, Smith B, Cahn PE, Ruiz-Palacios GM, Ikram A, Lanas F, Lourdes Guerrero M, Muñoz Navarro SR et al. (2022) Final efficacy analysis, interim safety analysis, and immunogenicity of a single dose of recombinant novel coronavirus vaccine (adenovirus type 5 vector) in adults 18 years and older: an international, multicentre, randomised, double-blinded, placebo-controlled phase 3 trial. Lancet 399:237-248. ).

After vaccines roll out in the real world, some safety concerns emerged in rare cases of adverse events. There is some evidence that intramuscular adenovirus application can induce thrombotic thrombocytopenia in susceptible individuals (McGonagle et al., 2021McGonagle D, de Marco G and Bridgewood C (2021) Mechanisms of Immunothrombosis in Vaccine-Induced Thrombotic Thrombocytopenia (VITT) Compared to Natural SARS-CoV-2 Infection. J Autoimmun 121:102662. ), and other similar blood disorders in rare cases after ChAd and Ad26 vaccination (Lundstrom et al., 2021Lundstrom K, Barh D, Uhal BD, Takayama K, Aljabali AAA, Abd El-Aziz TM, Lal A, Redwan EM, Adadi P, Chauhan G et al. (2021) COVID-19 Vaccines and Thrombosis-Roadblock or Dead-End Street? Biomolecules 11:1020. ; Sorensen et al., 2021Sorensen ALT, Rolland M, Hartmann J, Harboe ZB, Roed C, Jensen TO, Kolte L, Fassi D el, Hillingso J, Radziwon-Balicka A et al. (2021) A case of thrombocytopenia and multiple thromboses after vaccination with ChAdOx1 nCoV-19 against SARS-CoV-2. Blood Adv 5:2569-2574. ; Trogstad et al., 2021Trogstad L, Robertson AH, Mjaaland S and Magnus P (2021) Association between ChAdOx1 nCoV-19 vaccination and bleeding episodes: Large population-based cohort study. Vaccine 39:5854-5857. ; Walter et al., 2021Walter U, Fuchs M, Grossmann A, Walter M, Thiele T, Storch A and Wittstock M (2021) Adenovirus-Vectored COVID-19 Vaccine-Induced Immune Thrombosis of Carotid Artery. Neurology 97:716-719. ). This started a series of researches involving adenovirus modifications and changes in the route of application, like intranasal to overcome such events.

Adenovirus against tuberculosis and other bacteria

The adenovirus system has been tested and used against several other pathogens, not just for viruses. One example is tuberculosis (TB). For several years, alternative vaccines against TB have been studied, because the protection mediated by Bacillus Calmette-Guérin is not sufficient to control TB spread. Initially tested in mice models, recombinant adenoviral vaccines carrying immunogenic epitopes of Mycobacterium tuberculosis (AdAg85A) appeared to be a good option. It showed better immune protection administered intranasally when compared to BCG (Wang et al., 2004Wang J, Thorson L, Stokes RW, Santosuosso M, Huygen K, Zganiacz A, Hitt M and Xing Z (2004) Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis. J Immunol 173:6357-6365. ), it worked as a booster also for BCG prime alone (Santosuosso et al., 2006Santosuosso M, McCormick S, Zhang X, Zganiacz A and Xing Z (2006) Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis. Infect Immun 74:4634-4643. ; Li et al., 2015Li W, Li M, Deng G, Zhao L, Liu X and Wang Y (2015) Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice. Mol Med Rep 12:3073-3080), as well as when followed by modified Vaccinia virus Ankara vectors (You et al., 2012You Q, Wu Y, Wu Y, Wei W, Wang C, Jiang D, Yu X, Zhang X, Wang Y, Tang Z et al. (2012) Immunogenicity and protective efficacy of heterologous prime-boost regimens with mycobacterial vaccines and recombinant adenovirus- and poxvirus-vectored vaccines against murine tuberculosis. Int J Infect Dis 16:e816-25. ; Betts et al., 2012Betts G, Poyntz H, Stylianou E, Reyes-Sandoval A, Cottingham M, Hill A and McShane H (2012) Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection. PloS One 7:e50447. ; Stylianou et al., 2015Stylianou E, Griffiths KL, Poyntz HC, Harrington-Kandt R, Dicks MD, Stockdale L, Betts G and McShane H (2015) Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A. Vaccine 33:6800-6808. ; Kou et al., 2018Kou Y, Wan M, Shi W, Liu J, Zhao Z, Xu Y, Wei W, Sun B, Gao F, Cai L et al. (2018) Performance of Homologous and Heterologous Prime-Boost Immunization Regimens of Recombinant Adenovirus and Modified Vaccinia Virus Ankara Expressing an Ag85B-TB10.4 Fusion Protein against Mycobacterium tuberculosis. J Microbiol Biotechnol28:1022-1029. ).

Going further in other animal models that are susceptible to Mycobacterium infection, a recombinant adenovirus expressing multiple antigens (Ag85A, TB10.4, TB9.8 and Acr2) increased BCG protection after M. caprae challenge (Pérez De Val et al., 2013Pérez De Val B, Vidal E, Villarreal-Ramos B, Gilbert SC, Andaluz A, Moll X, Martín M, Nofrarías M, McShane H, Vordermeier HM et al. (2013) A multi-antigenic adenoviral-vectored vaccine improves BCG-induced protection of goats against pulmonary tuberculosis infection and prevents disease progression. PloS One 8:e81317. ); in guinea pigs after M. tuberculosis exposure, AdAg85A boost led to increased survival compared to BCG administration alone (Xing et al., 2009Xing Z, McFarland CT, Sallenave JM, Izzo A, Wang J and McMurray DN (2009) Intranasal Mucosal Boosting with an Adenovirus-Vectored Vaccine Markedly Enhances the Protection of BCG-Primed Guinea Pigs against Pulmonary Tuberculosis. PloS One 4:e5856. ). A similar result was observed in cattle using BCG as prime, AdAg85A as a booster and tested against M. bovis challenge (Dean et al., 2014Dean G, Whelan A, Clifford D, Salguero FJ, Xing Z, Gilbert S, McShane H, Hewinson RG, Vordermeier M and Villarreal-Ramos B (2014) Comparison of the immunogenicity and protection against bovine tuberculosis following immunization by BCG-priming and boosting with adenovirus or protein based vaccines. Vaccine 32:1304-1310. ). Interestingly, in rhesus macaques using BCG as prime and a boost of Ad5 vector caring TB antigens (M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) showed no enhanced protection against infection compared to BCG used alone (Darrah et al., 2019Darrah PA, DiFazio RM, Maiello P, Gideon HP, Myers AJ, Rodgers MA, Hackney JA, Lindenstrom T, Evans T, Scanga CA et al. (2019) Boosting BCG with proteins or rAd5 does not enhance protection against tuberculosis in rhesus macaques. NPJ vaccines4:21. ), and the same result was observed using a regimen of prime-boost strategy with ChAd3 and MVA (Vierboom et al., 2020Vierboom MPM, Chenine AL, Darrah PA, Vervenne RAW, Boot C, Hofman SO, Sombroek CC, Dijkman K, Khayum MA, Stammes MA et al. (2020) Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques. NPJ Vaccines 5:39. ).

Clinical trials using an Ad35 deficient vector carrying a fusion protein of three M. tuberculosis antigens (Ag85A, Ag85B and TB10.4) were performed on several target groups and showed to be safe in healthy volunteers (Hoft et al., 2012Hoft DF, Blazevic A, Stanley J, Landry B, Sizemore D, Kpamegan E, Gearhart J, Scott A, Kik S, Pau MG et al. (2012) A recombinant adenovirus expressing immunodominant TB antigens can significantly enhance BCG-induced human immunity. Vaccine 30:2098-2108. ; Sheehan et al., 2015Sheehan S, Harris SA, Satti I, Hokey DA, Dheenadhayalan V, Stockdale L, Manjaly Thomas ZR, Minhinnick A, Wilkie M, Vermaak S et al. (2015) A Phase I, Open-Label Trial, Evaluating the Safety and Immunogenicity of Candidate Tuberculosis Vaccines AERAS-402 and MVA85A, Administered by Prime-Boost Regime in BCG-Vaccinated Healthy Adults. PloS One 10:e0141687. ; Tameris et al., 2015Tameris M, Hokey DA, Nduba V, Sacarlal J, Laher F, Kiringa G, Gondo K, Lazarus EM, Gray GE, Nachman S et al. (2015) A double-blind, randomised, placebo-controlled, dose-finding trial of the novel tuberculosis vaccine AERAS-402, an adenovirus-vectored fusion protein, in healthy, BCG-vaccinated infants. Vaccine 33:2944-2954. ); infants 6-9 months (Kagina et al., 2014Kagina BMN, Tameris MD, Geldenhuys H, Hatherill M, Abel B, Hussey GD, Scriba TJ, Mahomed H, Sadoff JC, Hanekom WA et al. (2014) The novel tuberculosis vaccine, AERAS-402, is safe in healthy infants previously vaccinated with BCG, and induces dose-dependent CD4 and CD8T cell responses. Vaccine 32:5908-5917) and in subjects with TB latent infection (Walsh et al., 2016aWalsh DS, Owira V, Polhemus M, Otieno L, Andagalu B, Ogutu B, Waitumbi J, Hawkridge A, Shepherd B, Pau MG et al. (2016a) Adenovirus type 35-vectored tuberculosis vaccine has an acceptable safety and tolerability profile in healthy, BCG-vaccinated, QuantiFERON(®)-TB Gold (+) Kenyan adults without evidence of tuberculosis. Vaccine 34:2430-2436. ; van Zyl-Smit et al., 2017van Zyl-Smit RN, Esmail A, Bateman ME, Dawson R, Goldin J, van Rikxoort E, Douoguih M, Pau MG, Sadoff JC, McClain JB et al. (2017) Safety and Immunogenicity of Adenovirus 35 Tuberculosis Vaccine Candidate in Adults with Active or Previous Tuberculosis. A Randomized Trial. Am J Respir Crit Care Med 195:1171-1180. ). In addition, a phase 1b trial using Ad5-Ag85A in healthy volunteers showed higher levels of mucosal immune cells by aerosol administration than by muscle injection (Jeyanathan et al., 2022Jeyanathan M, Fritz DK, Afkhami S, Aguirre E, Howie KJ, Zganiacz A, Dvorkin-Gheva A, Thompson MR, Silver RF, Cusack RP et al. (2022) Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans. JCI Insight 7:e155655. ). No results about efficacy are available yet.

The adenoviral delivery system carrying bacterial proteins has also been used in the research of vaccines for other bacteria of medical importance. Against Bacillus anthracis, the agent of Anthrax, an Ad5 was constructed and tested via intranasal or intramuscular in mice and rabbits showing high survival rates after challenge (Tan et al., 2003Tan Y, Hackett NR, Boyer JL and Crystal RG (2003) Protective immunity evoked against anthrax lethal toxin after a single intramuscular administration of an adenovirus-based vaccine encoding humanized protective antigen. Hum Gene Ther 14:1673-1682. ; Kasuya et al., 2005Kasuya K, Boyer JL, Tan Y, Alipui DO, Hackett NR and Crystal RG (2005) Passive immunotherapy for anthrax toxin mediated by an adenovirus expressing an anti-protective antigen single-chain antibody. Mol Ther 11:237-244. ; McConnell et al., 2006McConnell MJ, Hanna PC and Imperiale MJ (2006) Cytokine response and survival of mice immunized with an adenovirus expressing Bacillus anthracis protective antigen domain 4. Infect Immun 74:1009-1015. ; Zhang et al., 2013aZhang J, Jex E, Feng T, Sivko GS, Baillie LW, Goldman S, van Kampen KR and Tang DCC (2013a) An Adenovirus-Vectored Nasal Vaccine Confers Rapid and Sustained Protection against Anthrax in a Single-Dose Regimen. Clin Vaccine Immunol 20:1-8. ; Wu et al., 2014Wu S, Zhang Z, Yu R, Zhang J, Liu Y, Song X, Yi S, Liu J, Chen J, Yin Y et al. (2014) Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity. Clin Vaccine Immunol 21:156-164. ; Krishnan et al., 2015Krishnan V, Andersen BH, Shoemaker C, Sivko GS, Tordoff KP, Stark GV, Zhang J, Feng T, Duchars M and Roberts MS (2015) Efficacy and immunogenicity of single-dose AdVAV intranasal anthrax vaccine compared to anthrax vaccine absorbed in an aerosolized spore rabbit challenge model. Clin Vaccine Immunol 22:430-439. ). Ad5 was used against Haemophilus influenza as well and tested in chinchillas (Winter and Barenkamp, 2010Winter LE and Barenkamp SJ (2010) Construction and immunogenicity of recombinant adenovirus vaccines expressing the HMW1, HMW2, or Hia adhesion protein of nontypeable Haemophilus influenzae. Clin Vaccine Immunol CVI 17:1567-1575. ), for Leptospira interrogans Ad5 was tested in gerbils (Branger et al., 2001Branger C, Sonrier C, Chatrenet B, Klonjkowski B, Ruvoen-Clouet N, Aubert A, André-Fontaine G and Eloit M (2001) Identification of the Hemolysis-Associated Protein 1 as a Cross-Protective Immunogen of Leptospira interrogans by Adenovirus-Mediated Vaccination. Infect Immun 69:6831. ), for Listeria monocytogenes (Christensen et al., 2013Christensen A, Randrup T, Anderschou H, Jensen D, Schlüter J, Pravsgaard S, Jensen MA and Steffensen B (2013) Adenovirus-Based Vaccine against Listeria monocytogenes: Extending the Concept of Invariant Chain Linkage. J Immunol 191:4152-4164. ), Pseudomonas aeruginosa (Worgall et al., 2005Worgall S, Krause A, Rivara M, Hee KK, Vintayen EV, Hackett NR, Roelvink PW, Bruder JT, Wickham TJ, Kovesdi I et al. (2005) Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid. J Clin Invest 115:1281. ) and Yersinia pestis (Kilgore et al., 2021Kilgore PB, Sha J, Andersson JA, Motin VL and Chopra AK (2021) A new generation needle- and adjuvant-free trivalent plague vaccine utilizing adenovirus-5 nanoparticle platform. NPJ Vaccines 6:21. ) the tests were done in mice, all showing immune response activation.

Prevention of other diseases

The possibilities for adenoviral vectors usage are endless, several other studies are underway for malaria (Ewer et al., 2015Ewer KJ, Sierra-Davidson K, Salman AM, Illingworth JJ, Draper SJ, Biswas S and Hill AVS (2015) Progress with viral vectored malaria vaccines: A multi-stage approach involving “unnatural immunity”. Vaccine 33:7444-7451. ; Hollingdale et al., 2017Hollingdale MR, Sedegah M and Limbach K (2017) Development of replication-deficient adenovirus malaria vaccines. Expert Rev Vaccines 16:261-271. ); Ebola (Matz et al., 2019Matz KM, Marzi A and Feldmann H (2019) Ebola vaccine trials: progress in vaccine safety and immunogenicity. Expert Rev Vaccines 18:1229-1242. ) and Marburg virus (Dulin et al., 2021Dulin N, Spanier A, Merino K, Hutter JN, Waterman PE, Lee C and Hamer MJ (2021) Systematic review of Marburg virus vaccine nonhuman primate studies and human clinical trials. Vaccine 39:202-208. ), Influenza virus (Kerstetter et al., 2021Kerstetter LJ, Buckley S, Bliss CM and Coughlan L (2021) Adenoviral Vectors as Vaccines for Emerging Avian Influenza Viruses. Front Immunol 11:607333. ), Dengue virus (Khanam et al., 2009Khanam S, Pilankatta R, Khanna N and Swaminathan S (2009) An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity. Vaccine27:6011-6021. ; George and Eo, 2011George JA and Eo SK (2011) Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2. Immune Netw 11:268. ), Chikungunya virus (Campos et al., 2019Campos RK, Preciado-Llanes L, Azar SR, Lopez-Camacho C, Reyes-Sandoval A and Rossi SL (2019) A Single and Un-Adjuvanted Dose of a Chimpanzee Adenovirus-Vectored Vaccine against Chikungunya Virus Fully Protects Mice from Lethal Disease. Pathogens 8:231.; Folegatti et al., 2021Folegatti PM, Harrison K, Preciado-Llanes L, Lopez FR, Bittaye M, Kim YC, Flaxman A, Bellamy D, Makinson R, Sheridan J et al. (2021) A single dose of ChAdOx1 Chik vaccine induces neutralizing antibodies against four chikungunya virus lineages in a phase 1 clinical trial. Nat Commun 12:4636. ) and Zika virus (Bullard et al., 2020Bullard BL, Corder BN, Gordon DN, Pierson TC and Weaver EA (2020) Characterization of a Species E Adenovirus Vector as a Zika virus vaccine. Sci Rep 10:3613. ; López-Camacho et al., 2020López-Camacho C, de Lorenzo G, Slon-Campos JL, Dowall S, Abbink P, Larocca RA, Kim YC, Poggianella M, Graham V, Findlay-Wilson S et al. (2020) Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines. Vaccines (Basel) 8:307. ), Hepatitis B virus (HBV) (Zhang et al., 2018bZhang X, Wang J, Lu J, Li R and Zhao S (2018b) Immunogenicity of adenovirus-vector vaccine targeting hepatitis B virus: Non-clinical safety assessment in non-human primates. Virol J 15:111. ; Chinnakannan et al., 2020Chinnakannan SK, Cargill TN, Donnison TA, Ansari MA, Sebastian S, Lee LN, Hutchings C, Klenerman P, Maini MK, Evans T et al. (2020) The Design and Development of a Multi-HBV Antigen Encoded in Chimpanzee Adenoviral and Modified Vaccinia Ankara Viral Vectors; A Novel Therapeutic Vaccine Strategy against HBV. Vaccines8:184. ), Hepatitis C virus (HCV) (Agrawal et al., 2019Agrawal B, Gupta N, Vedi S, Singh S, Li W, Garg S, Li J and Kumar R (2019) Heterologous Immunity between Adenoviruses and Hepatitis C Virus (HCV): Recombinant Adenovirus Vaccine Vectors Containing Antigens from Unrelated Pathogens Induce Cross-Reactive Immunity Against HCV Antigens. Cells 8:507. ; Hartnell et al., 2020Hartnell F, Esposito I, Swadling L, Brown A, Phetsouphanh C, de Lara C, Gentile C, Turner B, Dorrell L, Capone S et al. (2020) Characterizing Hepatitis C Virus-Specific CD4 + T Cells Following Viral-Vectored Vaccination, Directly Acting Antivirals, and Spontaneous Viral Cure. Hepatology 72:1541-1555. ), Human Respiratory Syncytial virus (HRSV) (Gomi et al., 2018Gomi R, Sharma A, Wu W and Worgall S (2018) Neonatal Genetic Delivery of Anti-Respiratory Syncytial Virus (RSV) Antibody by Non-Human Primate-Based Adenoviral Vector to Provide Protection against RSV. Vaccines7:3. ; Cicconi et al., 2020Cicconi P, Jones C, Sarkar E, Silva-Reyes L, Klenerman P, de Lara C, Hutchings C, Moris P, Janssens M, Fissette LA et al. (2020) First-in-Human Randomized Study to Assess the Safety and Immunogenicity of an Investigational Respiratory Syncytial Virus (RSV) Vaccine Based on Chimpanzee-Adenovirus-155 Viral Vector-Expressing RSV Fusion, Nucleocapsid, and Antitermination Viral Proteins in Healthy Adults. Clin Infect Dis 70:2073-2081. ; Williams et al., 2020Williams K, Bastian AR, Feldman RA, Omoruyi E, de Paepe E, Hendriks J, van Zeeburg H, Godeaux O, Langedijk JPM, Schuitemaker H et al. (2020) Phase 1 Safety and Immunogenicity Study of a Respiratory Syncytial Virus Vaccine With an Adenovirus 26 Vector Encoding Prefusion F (Ad26.RSV.preF) in Adults Aged ≥60 Years. J Infect Dis 222:979-988. ), Nipah virus (NiV) (van Doremalen et al., 2019van Doremalen N, Lambe T, Sebastian S, Bushmaker T, Fischer R, Feldmann F, Haddock E, Letko M, Avanzato VA, Rissanen I et al. (2019) A single-dose ChAdOx1-vectored vaccine provides complete protection against Nipah Bangladesh and Malaysia in Syrian golden hamsters. PLoS Negl Trop Dis 13:e0007462. ), Human Papillomavirus (HPV) (Li et al., 2016Li LL, Wang HR, Zhou ZY, Luo J, Xiao XQ, Wang XL, Li JT, Zhou YB and Zeng Y (2016) One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice. Antiviral Res 128:20-27. ; Wu et al., 2018Wu J, Chen G, Zhuang FC, Gao M, Wu CD, He ZL, Jiang YS, Li JB, Bao JY and Mao ZN (2018) Long-term toxicity, pharmacokinetics and immune effects of a recombinant adenovirus vaccine expressing human papillomavirus 16 E6 and E7 proteins (HPV16 E6E7-Ad5 Vac) in primates. Am J Transl Res 10:1539.), Rotavirus (Xie et al., 2015Xie L, Yan M, Wang X, Ye J, Mi K, Yan S, Niu X, Li H and Sun M (2015) Immunogenicity and efficacy in mice of an adenovirus-based bicistronic rotavirus vaccine expressing NSP4 and VP7. Virus Res 210:298-307. ) and many more. Adding to this, veterinary application, against pathogens like Foot-and-mouth disease virus (FMD) (Diaz-San Segundo et al., 2017Diaz-San Segundo F, Medina GN, Stenfeldt C, Arzt J and de los Santos T (2017) Foot-and-mouth disease vaccines. Vet Microbiol 206:102-112. ), Rift Valley fever virus (Stedman et al., 2019Stedman A, Wright D, Wichgers Schreur PJ, Clark MHA, Hill AVS, Gilbert SC, Francis MJ, van Keulen L, Kortekaas J, Charleston B et al. (2019) Safety and efficacy of ChAdOx1 RVF vaccine against Rift Valley fever in pregnant sheep and goats. NPJ Vaccines 4:44. ), Rabies virus (Wang et al., 2019bWang X, Fang Z, Xiong J, Yang K, Chi Y, Tang X, Ma L, Zhang R, Deng F, Lan K et al. (2019) A chimpanzee adenoviral vector-based rabies vaccine protects beagle dogs from lethal rabies virus challenge. Virology 536:32-38. ), Rabbit hemorrhagic disease virus (RHDV) (Jiang et al., 2018Jiang Q, Yu Z, Liu JS, Kong DS, Guo DC, Quan CS, Li BT, Hu XL and Qu L (2018) Recombinant canine adenovirus type 2 expressing rabbit hemorrhagic disease virus VP60 protein provided protection against RHD in rabbits. Vet Microbiol 213:15-20. ), African Swine Fever virus (Lokhandwala et al., 2017Lokhandwala S, Waghela SD, Bray J, Sangewar N, Charendoff C, Martin CL, Hassan WS, Koynarski T, Gabbert L, Burrage TG et al. (2017) Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine. PloS One 12:e0177007. ), Porcine Reproductive and Respiratory Syndrome virus (Zhu et al., 2014Zhu L, Song H, Zhang X, Xia X and Sun H (2014) Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors. Virol J 11:225.), Feline Immunodeficiency virus (Gonin et al., 1995Gonin P, Fournier A, Oualikene W, Moraillon A and Eloit M (1995) Immunization trial of cats with a replication-defective adenovirus type 5 expressing the ENV gene of feline immunodeficiency virus. Vet Microbiol 45:393-401. ) and much more. In addition, for Ebola, several formulations are in advanced clinical trials and some are already approved by many health regulatory agencies, including vaccines based on Ad26, Ad5 and ChAd3 (Woolsey and Geisbert, 2021Woolsey C and Geisbert TW (2021) Current state of Ebola virus vaccines: A snapshot. PLoS Pathog 17:e1010078.).

Modulation of immune system by adenovirus

One interesting aspect of adenovirus usage is the capability of immune modulation by choosing the inoculation route and by virus modifications. For example, mice immunized intraperitoneally with a replication-defective adenovirus elicited an IgGa immune response against the hexon, while intravenous application triggered an antibody isotype variation (Gahéry-Ségard et al., 1997Gahéry-Ségard H, Juillard V, Gaston J, Lengagne R, Pavirani A, Boulanger P and Guillet JG (1997) Humoral immune response to the capsid components of recombinant adenoviruses: routes of immunization modulate virus-induced Ig subclass shifts. Eur J Immunol 27:653-659. ). Adenoviral intranasal immunization induced higher levels of specific IgA on airway mucosa, higher systemic IgG1/IgG2a ratio and lower levels of IFN-γ secreting cells compared to subcutaneously application (Papp et al., 1999bPapp Z, Babiuk LA and Baca-Estrada ME (1999b) Antigen-specific cytokine and antibody isotype profiles induced by mucosal and systemic immunization with recombinant adenoviruses. Viral Immunol 12:107-116. ). Surprisingly, orally administered adenovirus, elicited systemic immune response rather than mucosal (Oomura et al., 2006Oomura K, Xin KQ, Takakura M, Shinoda K, Jounai N and Okuda K (2006) Oral administration of the adenovirus vector induces systemic immunity rather than intestinal mucosal immunity. Vaccine 24:1045-1046. ). Moreover, combination of different routes can help to obtain a stronger immune response. In a study model for HRSV, a regimen of oral prime and intranasal Ad vaccine boost was able to enhance immune response compared to each individually (Fu et al., 2010Fu YH, He JS, Wang XB, Zheng XX, Wu Q, Xie C, Zhang M, Wei W, Tang Q, Song JD et al. (2010) A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus. Biochem Biophys Res Commun 395:87-92. ).

Intramuscular injection generated high levels of CD8⁺ T cell, probably due to adenovirus’ ability to express considerable high quantities of antigens (Yang T et al., 2003Yang TC, Dayball K, Wan YH and Bramson J (2003) Detailed Analysis of the CD8+ T-Cell Response following Adenovirus Vaccination. J Virol 77:13407) plus its costimulatory effects in antigen-presenting cells (APCs), promoting activation and maturation of dendritic cells (DC) and inducing prolonged CD8⁺ T lymphocytes activation (Rea et al., 1999Rea D, Schagen FHE, Hoeben RC, Mehtali M, Havenga MJE, Toes REM, Melief CJM and Offringa R (1999) Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset. J Virol 73:10245-10253. ; Hensley et al., 2005Hensley SE, Giles-Davis W, McCoy KC, Weninger W and Ertl HCJ (2005) Dendritic cell maturation, but not CD8+ T cell induction, is dependent on type I IFN signaling during vaccination with adenovirus vectors. J Immunol 175:6032-6041. ; Tatsis et al., 2007bTatsis N, Fitzgerald JC, Reyes-Sandoval A, Harris-McCoy KC, Hensley SE, Zhou D, Lin SW, Bian A, Zhi QX, Iparraguirre A et al. (2007b) Adenoviral vectors persist in vivo and maintain activated CD8+ T cells: implications for their use as vaccines. Blood 110:1916. ). The same advantage of adenovirus to induce high expression levels can have a downside effect, this presentation for long periods can provoke T cells exhaustion, a state of dysfunctional role common in processes of chronic inflammation, fortunately, CD8⁺ T cells appeared to still work against virus challenge despite the exhausted phenotype (Yang et al., 2006Yang T-C, Millar J, Groves T, Grinshtein N, Parsons R, Takenaka S, Wan Y and Bramson JL (2006) The CD8+ T cell population elicited by recombinant adenovirus displays a novel partially exhausted phenotype associated with prolonged antigen presentation that nonetheless provides long-term immunity. J Immunol 176:200-210. ). There is a possibility that the exhausted T cells are related to the immunization route since systemic immunization induced impaired T cells but the same was not observed in the peripheral route (Holst et al., 2010Holst PJ, Ørskov C, Thomsen AR and Christensen JP (2010) Quality of the transgene-specific CD8+ T cell response induced by adenoviral vector immunization is critically influenced by virus dose and route of vaccination. J Immunol 184:4431-4439. ). Furthermore, in an HBV vaccination mice model, repeated vaccination with short intervals for a long period did not inhibit T cells induction, leaving a doubt if the exhausted phenotype is really impairing immune response (Boukhebza et al., 2014Boukhebza H, Dubois C, Koerper V, Evlachev A, Schlesinger Y, Menguy T, Silvestre N, Riedl P, Inchauspé G and Martin P (2014) Comparative analysis of immunization schedules using a novel adenovirus-based immunotherapeutic targeting hepatitis B in naïve and tolerant mouse models. Vaccine 32:3256-3263. ).

Another aspect of adenovirus infection and immune responses is controlled by the different receptors that each subtype preferably interacts with. Most Ad types use CAR to enter cells, others CD46, a receptor presented in DC cells that contribute to its infection and together with Toll Like Receptor 9 (TLR9) activation induces these cells to produce IFN-α (Iacobelli-Martinez and Nemerow, 2007Iacobelli-Martinez M and Nemerow GR (2007) Preferential activation of Toll-like receptor nine by CD46-utilizing adenoviruses. J Virol 81:1305-1312. ; Perreau et al., 2012Perreau M, Welles HC, Pellaton C, Gjoksi B, Potin L, Martin R, Harari A, Bett A, Casimiro D, Gall J et al. (2012) The number of Toll-like receptor 9-agonist motifs in the adenovirus genome correlates with induction of dendritic cell maturation by adenovirus immune complexes. J Virol 86:6279-6285. ). Therefore, the choice of adenovirus type is important. For example, Ad3 can be found in the liver and lung, while Ad37 in the spleen after intravenous administration; Ad3 and Ad4 can even be toxic for the liver (Appledorn et al., 2008Appledorn DM, Kiang A, McBride A, Jiang H, Seregin S, Scott JM, Stringer R, Kousa Y, Hoban M, Frank MM et al. (2008) Wild-type adenoviruses from groups A-F evoke unique innate immune responses, of which HAd3 and SAd23 are partially complement dependent. Gene Ther12:885-901. ). Additionally, responses can be type-specific, Ad28 and Ad35 are more efficient in infecting and activating DC cells than Ad5, but they also induce more IFN-α and that can reduce their effectiveness in vivo, which can be overcome by higher Ad doses to increase the duration of CD8⁺ T cells response (Johnson et al., 2012Johnson MJ, Petrovas C, Yamamoto T, Lindsay RWB, Loré K, Gall JGD, Gostick E, Lefebvre F, Cameron MJ, Price DA et al. (2012) Type I IFN induced by adenovirus serotypes 28 and 35 has multiple effects on T cell immunogenicity. J Immunol 188:6109-6118.).

Regarding innate immunity, it is important to emphasize that it is not a single TLR that is responsible for immune activation, apparently, multiple pathways are being activated by Ad, since knock out of each TLR individually did not change CD8⁺ response but absence of Myd88, an adaptor of TLR pathway, reduced it (Rhee et al., 2011Rhee EG, Blattman JN, Kasturi SP, Kelley RP, Kaufman DR, Lynch DM, la Porte A, Simmons NL, Clark SL, Pulendran B et al. (2011) Multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors. J Virol 85:315-323. ). In addition, specific activation of TLR4 is described as an important step to trigger an effective humoral immune response by Ad vectors (Li R et al., 2018Li R, Liu J, Wu S, Zai X, Li Y, Yang Q, Hou L, Xu J and Chen W (2018) Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality. Immunology 155:251-262. ). TLR4 agonists can also enhance activation of CD4⁺ and CD8⁺ T cells and pro-inflammatory cytokines when used as an adjuvant to Ad vaccination (Lebedeva et al., 2018Lebedeva E, Bagaev A, Pichugin A, Chulkina M, Lysenko A, Tutykhina I, Shmarov M, Logunov D, Naroditsky B and Ataullakhanov R (2018) The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector. BMC Immunol 19:26. ).

Cytokine production has different modulatory activities depending on Ad type; Ad26, Ad35 and Ad48 use CD46 receptor induce more IFN-γ, 10-kDa gamma interferon-induced protein (IP-10), interleukin 1 receptor antagonist (IL-1RA) and IL-6, all related to a proinflammatory pattern, compared to Ad5, a type that uses CAR receptor (Teigler et al., 2012Teigler JE, Iampietro MJ and Barouch DH (2012) Vaccination with adenovirus serotypes 35, 26, and 48 elicits higher levels of innate cytokine responses than adenovirus serotype 5 in rhesus monkeys. J Virol 86:9590-9598).

An additional layer to consider when using adenoviral vectors is related to previous immune responses to the specific type used, since prior exposure to the Ad can interfere with its capability to induce an immune response, especially in homologous prime-boost regimens (Yang Z et al., 2003Yang Z, Wyatt LS, Kong W, Moodie Z, Moss B and Nabel GJ (2003) Overcoming immunity to a viral vaccine by DNA priming before vector boosting. J Virol 77:799-803. ; Schuldt et al., 2012Schuldt NJ, Aldhamen YA, Godbehere-Roosa S, Seregin SS, Kousa YA and Amalfitano A (2012) Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in nave and Adenovirus (Ad5) immune mice. Malar J 11:209.). However, usage of different types of adenoviruses can overcome this problem, as exemplified by researchers’ experiments with isotypes of defective chimpanzee adenovirus applied in a heterologous prime-boost immunization; they showed induction of a high frequency of specific CD8⁺ T cells (Pinto et al., 2003Pinto AR, Fitzgerald JC, Giles-Davis W, Gao GP, Wilson JM and Ertl HCJ (2003) Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers. J Immunol 171:6774-6779. ). Moreover, usage of rare Ad types can contribute to activation of phenotypically different T cells triggering polyfunctional immune responses (Liu et al., 2008Liu J, Ewald BA, Lynch DM, Denholtz M, Abbink P, Lemckert AAC, Carville A, Mansfield KG, Havenga MJ, Goudsmit J et al. (2008) Magnitude and Phenotype of Cellular Immune Responses Elicited by Recombinant Adenovirus Vectors and Heterologous Prime-Boost Regimens in Rhesus Monkeys. J Virol 82:4844-4852. ). In conclusion, to use the full potential of Ad vectors, all aspects of their interaction with the immune system need to be evaluated and extensively studied, especially considering the different applications in gene therapy, cancer treatment and vaccine development.

Conclusion

Adenovirus is a double-stranded DNA virus that does not integrate the host genome, remaining episomal. Gene transfer mediated by adenoviral vectors is not sustained for long periods, unless it has been altered to be able to integrate. This limits its application for monogenic diseases treatment. However, for cancer gene therapy, transgene expression has to last only long enough to mediate the tumor cells death, the choice of the transgene has to take into account the tumor cell and microenvironment, limiting its nutritional supply, preventing proliferation, inducing cell death and recruiting and activating immune cells capable of destroying the tumor cells and averting dissemination to other sites. The recent and broad use of adenoviral vectors as vaccination tools in the COVID-19 crisis has put this technology in the spotlight and overall, it had success, there are some issues to be solved and questions to be answered, like if the individuals vaccinated with adenoviral vectors will develop neutralizing antibodies that will impede its future use. In Figure 3 we discuss the distribution of clinical trials involving adenoviral technology. It has been a long and bumpy road along the way. But the continuous effort in this research field may warrant new successful therapies and vaccines.

Acknowledgments

Financial support was received from the São Paulo Research Foundation, FAPESP (RET: 2019/15619-2, 2020/08271-7). Fellowships were received from FAPESP to NMA: 21/03418-2 and NPAT: 21/02659-6)

References

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Supplementary material

The following online material is available for this article:

Table S1 - Adenoviral vectors modulating proliferation and survival of tumor cells.

Table S2 - Adenoviral vectors inducing growth suppressors.

Table S3 - Adenoviral vectors inducing cell death.

Table S4 - Adenoviral vectors modulating tumoral angiogenesis.

Table S5 - Adenoviral vectors modulating invasion and metastasis.

Table S6 - Adenoviral vectors modulating immune system.

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