AAV-MEDIATED, IN VIVO GENE DELIVERY TO THE ROTATOR CUFF

Tendon injuries present a considerable clinical challenge due to the limited regenerative capacity of tendons. The use of gene transfer to deliver growth factors to sites of tendon damage has been suggested as a promising strategy for improving tendon regeneration. A major issue for this approach is to identify clinically acceptable vectors that can deliver genes to the cells of the tendon, preferably by in vivo delivery. Adeno-associated virus (AAV) has many advantages in this regard, including a favourable safety profile and the ability to sustain long-term transgene expression. Here we explored the use of AAV to deliver marker genes to the supra-and infra-spinatus tendons of the rotator cuff in the rat by injection into the subacromial space. First, we screened various AAV serotypes for their transducing ability towards rat and human tenocytes in vitro . Of the 10 serotypes tested, AAV2.5 and AAV2 exhibited the highest in vitro transduction efficiency in both rat and human tenocytes. Ex vivo transduction of cells within explants of isolated, intact tendon was also demonstrated. Injection of AAV2.5 encoding luciferase into the subacromial space confirmed gene delivery to the infra-, but not supra-, spinatus tendon in vivo with transgene expression persisting for 7 days post-transduction. These data demonstrate the ability of AAV2.5 to deliver genes to the infraspinatus tendon, leading to sustained local expression following in vivo delivery. Our findings suggest that AAV2.5 has several advantages as vector for stimulating tendon regeneration by local, in vivo , gene transfer.

. These factors can lead to structural changes in the tendon, resulting in degeneration, inflammation and tears that cause pain, functional limitations and impaired quality of life (Ackerman et al., 2021;Docheva et al., 2015).
Tenocytes are specialized fibroblast-like cells that play a crucial role in the structure and function of tendons (Kannus, 2000;Li et al., 2021).Scleraxis and tenomodulin are specific molecular markers of tenocytes that are likely to play major roles in tendon regeneration (Aslan et al., 2008).
Tenocytes produce extracellular matrix proteins including collagen types I and III, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs), which are involved in the healing and remodeling processes in the tendon (Birch et al., 2013;Subramanian and Schilling, 2015).
Rotator cuff tendons, forearm extensors, Achilles tendon, tibialis posterior and patellar tendons are particularly prone to injury.Shoulder disorders account for over 4.5 million patient visits annually in the United States (Gomoll et al., 2004;Linaker and Walker-Bone, 2015).Indeed, injuries to the rotator cuff are among the most common musculotendinous conditions treated by orthopaedic surgeons.The rotator cuff comprises multiple tendons; healing of damaged tendons is typically slow and often leads to suboptimal outcomes in terms of structural integrity and mechanical strength (Dang and Davies, 2018).This poses a significant clinical challenge with a considerable impact on the well-being of patients, highlighting the need for effective treatment strategies to address tendon injuries and alleviate the associated burden (Jain et al., 2013;Teunis et al., 2014).
Various growth factors show promise as mediators of tendon regeneration (Docheva et al., 2015) but they are difficult to deliver in a sustained fashion to sites of tendon damage.Gene delivery has been suggested as a means to overcome the delivery problem but little progress has been made in applying this concept to tendons; there have been few large animal studies and no clinical trials (Evans and Huard, 2015).Selection of the vector to use is a critical preclinical decision when developing a translational gene therapy programme.Efficacy, safety and cost are all key factors; for a common, non-lethal condition such as tendon injury; safety and cost are particularly important.Vector deployment strategies also need careful consideration, with in vivo gene delivery being much more practical and less expensive than ex vivo protocols that require the expansion of autologous cells (Evans et al., 2021;Evans et al., 2007).

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In this context, adeno-associated virus (AAV) is an attractive vector.It is widely utilized in human gene therapy applications due to its safety, broad tropism and low immunogenicity (Evans and Huard, 2015;Greelish et al., 1999).This vector possesses the ability to transduce both dividing and nondividing cells while demonstrating limited cytotoxicity; it rarely integrates into the host chromosomes yet can produce long-term expression of the transgene (Hastie and Samulski, 2015;Li and Samulski, 2020).Several AAV-based gene therapies have received marketing approval from the U.S. Food and Drug Administration (FDA).
In the present study, we investigated the in vitro transduction efficiency of AAV vectors of different serotypes using tenocytes derived from rat supraspinatus and human Achilles tendons and used the most potent of these to deliver a marker gene to the rat rotator cuff in vivo.Because transduction efficiency in vitro is not always predictive of transduction efficiency in vivo, we undertook additional studies in the rat aimed at determining the ability of AAV2.5 to transduce cells within the rotator cuff in vivo.This also allowed us to measure the duration of transgene expression in vivo and to investigate the localization of transgene expression.

Cell culture
This study investigated transduction of the supraand infra-spinatus tendons of the rotator cuff through in vitro, ex vivo, and in vivo approaches.For in vitro studies, tenocytes were isolated from two male F344 rats, aged between 5 and 7 months.
Animal studies were conducted following the guidelines of Mayo Clinic's Institutional Animal Care and Use Committee (Protocol Number: A00004279).Euthanasia of the animals was carried out in accordance with the institutional humane euthanasia policy, using carbon dioxide inhalation.Immediately after confirming expiry, tenocytes were isolated from harvested supraspinatus tendon.Briefly, the tissue samples were washed multiple times with phosphatebuffered saline (PBS) (Gibco, Gaithersburg, MD, USA) containing 1 % penicillin/streptomycin (P/S) (Gibco).The supraspinatus tendons were then dissected into small pieces (1-2 mm) and incubated in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 5 % foetal bovine serum (FBS) (Gibco), 1 % P/S and 0.25 % w/v collagenase type II (Gibco) for 2-3 hours at 37 °C.Following digestion, the tendon fragments were filtered through a 100 µ m cell strainer and centrifuged at 400 g for 5 minutes.The cells were transferred to a tissue culture flask at a density of 2 × 10 4 cells/cm 2 and cultured in growth medium (high-glucose DMEM supplemented with 2 % L-glutamine, 10 % FBS, and 1 % P/S).Cells were maintained at 37 °C, 95 % humidity, and 5 % carbon dioxide, with a medium change every 2-3 days.Once the cultured cells reached 80-90 % confluence, they were detached with TrypLE™ (Thermo Fischer Scientific, Waltham, MA, USA) and subcultured at a density of 1 × 10 4 cells/cm 2 until passage 4-5.
Human tenocytes isolated from Achilles tendon were purchased from ZenBio (ZenBio Inc., Durham, NC, USA; Cat No # TEN-F; Lot No TEN012121A).According to the suppliers, the cells expressed types I and III collagen, thrombospondin 4 and scleraxis, as well as CD44 and CD90.They were negative for CD45 and CD31.Cells were expanded in Tenocyte Growth Medium (ZenBio Inc.; Cat No # TEN-F1) supplemented with 10 % MSC-grade FBS (Takara Bio, San Jose, CA, USA), and 1 % P/S (Gibco) following the manufacturer's instructions (ZenBio Inc.; instruction manual zbm0075.04).For all experiments, human tenocytes at passage 2-4 were utilised.2, 2.5, 3, 4, 5, 6, 8, 9, rh10), and the selfcomplementary transgene plasmid encoding a green fluorescent protein (GFP)-luciferase fusion protein reporter with gene expression driven by a cytomegalovirus (CMV) promoter.Twenty-four hours post-transfection the medium (low glucose DMEM, 10 % FBS, 1 % P/S) was changed, and the cells harvested 72 hours post-transfection.Following three rounds of freeze-thaw with dry ice and an ethanol bath, the resulting lysate was added to an iodixanol density column and ultracentrifuged at 62,500 g for 2 hours.The fraction of the iodixanol gradient containing the 40 % density layer was pulled and was diluted with PBS prior to concentration via 100 kDa Amicon filters (Millipore Sigma, St. Louis, MO, USA).Once concentrated, the AAV was titrated using quantitative polymerase chain reaction (qPCR) with Sybr Green and primers targeting the inverted terminal repeat (ITR), (forward: ITR primer, 5′-GGAACCCCTAGTGATGGAGTT, reverse: ITR primer, 5′-CGGCCTCAGTGAGCGA).

Generation of AAV serotypes
For ex vivo tissue explant culture experiments and for in vivo experiments, AAV2.5 vectors expressing GFP or luciferase were obtained from a commercial source (Welgen Inc., Worcester, MA, USA).

Transduction of tenocytes with AAV
A total of 10,000 rat or human cells were seeded into each well of a 96-well plate, with 100 μL of growth medium.The next day, the cells were transduced with AAV serotypes to be tested (1, 2, 2.5, 3, 4, 5, 6, 8, 9, rh10) at a concentration of 1 × 10 4 vector genome copies (vg) in 25 μL of serumfree DMEM.After a 2-hour incubation period, an additional 75 μL of growth medium were added to each well.Following overnight transduction, the medium containing the virus was replaced with fresh growth medium.Cells were harvested 72 hours post-transduction.

Transduction efficiency
To assess the transduction efficiency of individual AAV serotypes, the number of GFP+ cells were counted in three different microscopic fields within a defined region of interest (ROI), Counting was performed in triplicate samples for each serotype and the average number of GFP+ cells per field was determined.Fluorescence microscopy (Olympus, Center Valley, PA, USA) at 10× magnification was used to visualize and count.

Firefly luciferase activity
Luciferase activity in the cell lysates was determined following the instructions provided in the user's manual (Promega, Madison, WI, USA).Briefly, 100 µ L of the Glo-lysis buffer were used to lyse the cells.Subsequently, 30 µ L of the cell lysate were transferred to 96-well plates in triplicate.Then, 30 µ L Luciferase Assay Reagent (Bright-Glo™ Luciferase assay system, Promega) were added to each well, and the plates were incubated for 10 minutes at room temperature.Firefly luminescence was measured using a microplate luminometer (Varioskan Lux, Thermo Fisher Scientific).

Picogreen DNA assay
The total DNA content of the cell lysate was quantified using the QuantiT PicoGreen doublestranded DNA (dsDNA) Assay Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions.To generate a standard curve for quantification, a series of DNA standard solutions was prepared.In a black 96well plate, 100 µ L of each sample at a dilution of 1:10, DNA standards and a blank control were transferred to wells.Then, 100 µ L of the picogreen assay reagent were added to each well.The plate was incubated for 5 minutes to allow the picogreen dye to bind to the DNA.The fluorescence intensity was measured using a fluorescence plate reader (Varioskan Lux, Thermo Fisher Scientific) at the excitation wavelength of 485 nm and emission wavelength of 535 nm.The resulting fluorescence measurements were used to determine the amount of DNA in each sample, and the luciferase activity data were normalised to nanogrammes of DNA.

Transduction of explanted tissue
The supraspinatus tendon of one rat, including paratenon, and muscle, was carefully isolated from the surrounding tissue.The isolated tissue was then thoroughly washed with sterile PBS to eliminate debris and remove any residual blood.www.ecmjournal.orgNext, the tendon was transferred to ultra-low attachment 96-well plates.For AAV2.5-GFP (Welgen Inc.) transduction, 5 × 10 9 vg were added to each well in 50 μL of serum-free DMEM.To enhance transduction efficiency, spinoculation was performed at 2000 g for 20 minutes at 37 °C.Following a 2-hour incubation period, an additional 100 µ L of growth medium were added to each well.After overnight transduction, the medium was replaced with fresh growth medium.At 48 hours post-infection, the tissue explants were fixed for subsequent immunohistochemical analysis.

Immunohistochemical staining
Tendons were fixed in 10 % neutral buffered formalin for 48 hours (n = 1).Fixed specimens were dehydrated in a series of graded ethanols and embedded in paraffin.5-μm sections were cut using an automatic microtome (HM 355S, Thermo Fischer Scientific) and mounted onto positively charged slides (Superfrost™ Plus Microscope Slides, Thermo Fisher Scientific).Expression of GFP was detected by enzymatic immunohistochemistry following a previously published protocol (De La Vega et al., 2018).Briefly, the formalin-fixed tissue sections were deparaffinised, rehydrated, and washed twice in PBS for 5 minutes.Endogenous peroxidase activity was blocked with peroxidase blocking reagent (Vectastain® Elite® , Vector Laboratories, Newark, CA, USA; Cat no # PK-6105).Nonspecific binding was blocked with 5 % rabbit serum.Subsequently, the slides were incubated overnight at 4 °C with a rabbit polyclonal anti-GFP antibody (Abcam, Boston, MA, USA; Cat no # Ab27478) at a dilution of 1:1000.On the following day, slides were washed with PBS, and incubated with biotinylated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (Vector Laboratories; Cat no # BA-1000), followed by detection with an avidin-biotin-based peroxidase kit (Vectastain Elite ABC HRP Kit™; Vector laboratories).Slides were incubated in 3,3′ diaminobenzidine (ImmPACT DAB, Vector Laboratories), a peroxidase substrate that yields a brown product, followed by counterstaining with haematoxylin.Positive controls were formalinfixed, paraffin-embedded femora from transgenic rats expressing GFP constitutively (De La Vega et al., 2018).Non-transduced tendon was used as negative control and an isotype control for anti-GFP antibody (Abcam; Cat no # Ab240) was used to distinguish non-specific background signal from GFP signal.The stained sections were visualised using an Olympus BX43 microscope equipped with an Olympus SC50 camera (Olympus, Center Valley, PA, USA).
The same protocol was used for immunohistochemical staining of luciferase (n = 2), but in this case an anti-luciferase antibody (Novus Biologics, Minneapolis, MN, USA; Cat no # NB100-1677) at a dilution of 1:1000 was used as the primary antibody.Rabbit anti-goat IgG antibody (Novus Biologics; Cat no # NBP1-74526) was employed as the secondary antibody.
In vivo studies Surgery Animal care protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Mayo Clinic (A00007034-23).
Surgical procedures were conducted on a total of five skeletally mature male Fischer 344 rats, 8 months old, weighing between 300 and 450 g (Charles River Laboratories, Wilmington, MA, USA).Rats were housed in Mayo Clinic animal care facility with 12-hour light cycles and were given chow and water ad libitum.Prior to surgery, all animals received a pre-operative subcutaneous injection of 1 mg/kg buprenorphine-ER 0.5 mg/mL analgesic for pain management.Anaesthesia was induced using inhaled isoflurane at a concentration of 1-3 %.Both shoulders were shaved and sterilised using 70 % ethanol and iodopovidone.Under sterile conditions and with continuous anaesthesia using inhaled isoflurane, a 1-2 cm incision was made on the lateral right shoulder, proximal to the clavicle to expose the shoulder region.Injections were performed using a 25 µ L Hamilton® syringe (Hamilton, Reno, Nevada, USA; Cat no # 7636-01; 25 µ L, Model 702 RN SYR) with a 28-gauge needle, under direct visualisation into the subacromial space above the supraspinatus tendon.Correct subacromial positioning was confirmed visually before injecting the vector.No muscles were detached.Since the injection was performed into subacromial space, it is likely that the bursa was also exposed to the virus, but we did not verify this. www.ecmjournal.org The right shoulder was injected in the subacromial space with AAV2.5 encoding luciferase (Welgen Inc.), consisting of 1 × 10 8 vg/µ L suspended in 15 μL of sterile PBS (Thermo Fisher Scientific).The left shoulder was prepped and injected in an identical fashion to the right shoulder but received an injection of sterile PBS alone.The cutaneous skin incision was closed using continuous sutures technique with a 4-0 Vicryl suture (Ethicon™, Inc., Somerville, NJ, USA).Two additional rats underwent this survival procedure, receiving the same concentration of virus in the right shoulder, and sterile PBS for control in the left shoulder.A fourth rat underwent this surgery receiving virus in both shoulders, and a fifth rat underwent this surgery receiving sterile PBS in both shoulders.Rats were injected this way to be as efficient as possible in acquiring adequate samples for both imaging and histology.

Bioluminescence imaging
Two rats injected with AAV2.5 encoding luciferase were subjected to bioluminescence imaging using an in vivo imaging system (IVIS) spectrum (PerkinElmer Health Sciences Inc., Waltham, MA, USA) at designated time points following the surgical procedure.Rats were anaesthetised with 1-3 % isoflurane and administered an intraperitoneal injection of D-Luciferin (GoldBio, St. Louis, MO, USA) diluted in 15 mg/mL of PBS at a final dose of 150 mg/kg.Rats were positioned within the IVIS under continued isoflurane anaesthesia (2 %-2.5 % in 1 L/min O2) and imaged 20 minutes after Dluciferin delivery.The resulting bioluminescence signal was superimposed onto a photograph of the rat, and signal intensity was quantified using Living Image software 4.7.2 (PerkinElmer Health Sciences Inc).Immediately after imaging, the rat selected for additional, ex vivo experimentation was euthanised using 3.5-4 L/min of CO2 for 5 minutes.Both shoulders were harvested while isolating the supraspinatus and infraspinatus tendons, as well as adjacent cartilage, muscle and bone, which were placed into individual wells in a 12-well plate containing D-luciferin (300 µ g/mL).IVIS images were taken and the resulting bioluminescence signal (1 min, 8 min) superimposed onto a photograph of the tissue within the well-plate.

Statistical analyses
All statistical analyses were performed using GraphPad Prism 9.2.0 (GraphPad, Boston, MA, USA).There were 3 replicates (n = 3) for each experiment.All data are represented as average ± SD, as indicated in the figures.The Kolmogorov-Smirnov with Dallal-Wilkinson-Liliefor p-value and Shapiro-Wilk tests were performed to determine the data distribution.For normally distributed data, an unpaired t-test with Welch's correction was used to compare two independent groups.In multiple comparisons, one-way-ANOVA followed by Tukey's post hoc test was used.For non-normally distributed data, Mann-Whitney U-test was used to compare two independent samples.A p-value < 0.05 was considered statistically significant.

AAV2.5 demonstrated highest transduction efficiency in both rat and human tenocytes
A GFP-luciferase fusion protein under the transcriptional control of the CMV promoter was used to examine the tropism of the ten different scAAV serotypes listed in the methods section.Table 1 shows the titres that were obtained for each of these serotypes.When observed by fluorescence microscopy, cultures of rat tenocytes transduced by AAV2 and AAV2.5 contained the greatest number of GFP+ cells (Fig. 1A,B).Other serotypes, in contrast, had little or no transducing ability.Measurement of GFP fluorescence suggested that AAV2.5 produced greater GFP expression than AAV2, with AAV6, AAV9 and AAVrh10 producing low levels of GFP expression that were not seen by eye (Fig. 1C) (**p < 0.01; ***p < 0.001).Measurement of luciferase activity also suggested superior transgene expression using AAV2.5 (Fig. 1D) (*p < 0.05).Similar results were obtained for the human tenocytes (Fig. 2).Based on GFP expression, AAV2 and AAV2.5 had approximately equal transducing potency and were clearly superior to other serotypes (Fig. 2A,B,C).As with the rat tenocytes, when measuring luciferase activity AAV2.5 was superior to AAV2 and other serotypes (Fig. 2D) (*p < 0.05; **p < 0.01).
On the basis of these results, we selected AAV2.5 to take forward into subsequent ex vivo and in vivo experiments.

Ex vivo AAV2.5 transduction of the rat supraspinatus revealed the presence of GFPpositive cells not only in tenocytes but also in the paratenon and surrounding muscle
Intact supraspinatus tendon was dissected from rat shoulders and transduced in organ culture with AAV2.5.GFP to confirm transduction cells in situ and to locate transduced cells.GFP fluorescence imaging (Fig. 3A) demonstrated successful transduction of tenocytes, with a notable presence of GFP-positive cells in the paratenon (Fig. 3B).
Additionally, immunohistochemical analysis confirmed the presence of GFP-positive cells within the tendon, paratenon, and as well as the adjacent muscle (Fig. 3C).

Rat infraspinatus efficiently transduced by AAV2.5 in vivo
To confirm transduction in vivo, AAV2.5 luciferase was injected into the subacromial space of the shoulders of rats.Transgene expression was then analysed using an in vivo live imaging system.After a 48-hour delay, robust luciferase expression was visualized until day 7 (Fig. 4A).Highest luminescence intensity occurred on day 6 (Fig. 4B).Luciferase expression was limited to the subacromial space of the shoulder.Immunohistochemical analysis from this in vivo study demonstrated luciferase expression in rotator cuff (Fig. 4C).In a subsequent experiment, each component of the rotator cuff from the in vivo study was individually dissected and subjected to IVIS analysis.Ex vivo bioluminescence imaging localised transgene expression to the infraspinatus tendon with no signal observed from the supraspinatus tendon (Fig. 4D,E,F), (**p < 0.01), cartilage, muscle or bone (data not shown).Subsequent, immunohistochemistry analysis confirmed transgene expression in rat infraspinatus (Fig. 4G).Luciferase (n = 1).Boxed images show in vivo expression of luciferase.(B) Quantification analysis of luminescent intensity in AAV2.5-Luciferase injected rats for up to 8 days.(C) Immunohistochemistry staining of luciferase activity in infraspinatus tendon at day 6 (n = 1).(D) IVIS image of peak luciferase expression at day 6, compared to control (n = 3).(E) Quantification of IVIS imaging at day 6.(F) Ex vivo live imaging and (G) immunohistochemistry analysis of luciferase activity in the supra-and infraspinatus tendons harvested from AAV2.5-Luciferase injected shoulders at day 6.Scale bar, 50 µ m.Quantitative data are presented as mean ± SD. **p < 0.01.

Discussion
The concept of using gene delivery to promote tendon healing and regeneration is well established and both non-viral (Jin et al., 2022) and viral vectors have been studied for this purpose (Docheva et al., 2015).Although some progress has been reported with non-viral vectors (Jin et al., 2022) transfection efficiency is poor and transgene expression is low and transient.Viral vectors (Watson-Levings et al., 2022) are much more effective in gene transfer but are more complicated and expensive to manufacture and raise safety concerns.Because tendon injuries are not life-threatening, safety is a major issue that rules out the use of integrating vectors such as retrovirus and lentivirus.Adenovirus has been the most studied as a vector for gene therapy to tendon cells using a variety of different transgenes (Haddad-Weber et al., 2010;Lou et al., 2001).While the data are encouraging, no progress has been made in clinical translation.Among the disadvantages of adenoviral vectors is their antigenicity, especially with regard to activation of the innate immune system and consequent inflammation which is inimical to healing (Watson-Levings et al., 2022).In this context AAV offers several advantages as a vector.It is considered safer than other viral vectors because the wild-type virus causes no known disease.Although AAV generates a humoral, serotype-specific immune response, it does not typically activate cell-mediated responses.Several AAV-based gene therapies have been approved by the regulatory authorities and AAV has been approved for use by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA); AAV is in clinical trials of gene therapies for osteoarthritis, a non-lethal disease (Evans et al., 2023).
AAV exists in a number of different natural and artefactual serotypes with different tropisms (Issa et al., 2023).In the present study we compared the abilities of several of these serotypes to transduce tendon cells in vitro, ex vivo and in vivo.The data confirm the ability of AAV2 and, especially, AAV2.5 to transduce tenocytes within the rat infraspinatus tendon in vitro, ex vivo and in vivo, and to transduce human tenocytes in vitro.Other serotypes were less effective or ineffective.The relative potencies of AAV2 and AAV2.5 were not always consistent between the two reporter sequences.The marker gene encodes a GFP-luciferase fusion protein, so the discrepancies cannot reflect transduction efficiency or transgene expression.GFP has a longer half-life than luciferase which may account for the differences in expression recorded in Fig. 1C,D and Fig. 2C,D.Alternatively, differences in the measurement methodsfluorescence microscopy for GFP and enzyme assay for luciferase-may well account for this.
The effectiveness of AAV as a vector for in vivo delivery is a major practical advantage for future clinical development, and the expression period of 7 days may be appropriate for triggering a lasting regenerative response.AAV2.5 is a recently developed serotype created by incorporating 5 amino acids from AAV1 into the capsid protein of AAV2.This modification was primarily intended to decrease susceptibility to neutralizing antibodies that specifically target AAV2 which are present in approximately 30-60 % of the general population due to previous AAV2 infections (Abdul et al., 2023;Calcedo and Wilson, 2013).AAV2.5 has been used in clinical trials for muscular dystrophy (Bowles et al., 2012) and osteoarthritis (ClinicalTrials.govIdentifier: NCT02790723).Our study further demonstrated the potential of AAV2.5 in enhancing the transduction ability for the rotator cuff.
The previous literature on the use of AAV for tendon healing is sparse.Only a few studies have investigated the feasibility of gene transfer in tendons.Various serotypes of AAV exhibit differences in their tropism for target cells (Issa et www.ecmjournal.org al., 2023).AAV2, in particular, has been extensively studied and demonstrates a broad infectivity profile.For example, Wang et al. (2007) also found that AAV2 delivered exogenous genes to cultures of rat intrasynovial tenocytes, whereas AAV1, AAV3, AAV4, AAV5, AAV7, and AAV8 were ineffective.AAV serotype 2.5 was not tested.When AAV2 was used to transfer basic fibroblast growth factor (b-FGF) to tenocytes in cell culture, the investigators observed a significant increase in the expression of type I and III collagen, consistent with a regenerative response (Wang et al., 2005).The same group went on to construct AAV2 encoding a microRNA that silenced transforming growth factor beta 1 (TGF-β1).This vector suppressed adhesion formation in a chicken model of flexor tendon healing, when injected into the injured tendons intra-operatively (Wu et al., 2016).In later work they used AAV2 to deliver vascular endothelial growth factor (VEGF) in the same chicken flexor tendon healing model and noted increased strength of the healing tendon (Mao et al., 2017).In a different approach to AAVmediated tendon healing, Basile et al. ( 2008) coated AAV2 encoding growth/differentiation factor 5 (GDF-5) onto freeze-dried murine digital flexor tendon and implanted the construct in a model of flexor tendon healing.Here the intent was not to transduce tenocytes, because the allograft lacked living cells.Instead, after implantation the vector transduced cells surrounding the defect which secreted GDF-5 locally where it enhanced the healing process.In a subsequent study this group used AAV2.5 expressing GDF-5 in the freeze-dried allograft method, producing results that were broadly similar to those obtained with AAV2 (Hasslund et al., 2014).
We found that AAV2.5 preferentially transduced cells within the paratenon region surrounding the supraspinatus, compared to other areas of the tendon.Some transduction of cells was also observed in the adjacent supraspinatus muscle.The paratenon region of the tendon is more cellular than the main body of the tendon (López De Padilla et al., 2021) and plays a role in providing blood vessels and progenitor cells to the tendon (Müller et al., 2018).While the exact function of the paratenon in tendon healing is not fully understood, lineage tracing studies in mice have demonstrated the presence of a population of progenitor cells within the paratenon expressing smooth muscle actin, which proliferate during injury and contribute to the healing process of injured tendons (Dyment et al., 2014).Previous research by Müller et al. (2018) demonstrated an important role for the paratenon in healing an Achilles tendon defect model in rats.The ability of AAV2.5 to transduce cells within the paratenon may therefore be of high significance.Additional cell types are likely to be present in the setting of a tendon injury, but it is not possible to predict whether these will be transduced by AAV2.5.In addition to tenocytes, AAV2.5 is known to transduce chondrocytes, synovial fibroblasts (Watson Levings et al., 2018) and, as confirmed here, muscle cells.
Although AAV2.5 was found to transduce cells within both the infra-and supra-spinatus tendons of explanted rat tissue, only the infraspinatus tendon was transduced following in vivo injection into the subacromial space.The explanation for this presumably resides with the anatomy of the rat shoulder (Edelstein et al., 2011).The supraspinatus tendon sits more anteriorly in the subacromial space, so it may be partially shielded or less accessible to vectors injected into this area (Andarawis-Puri et al., 2009).Thus, the infraspinatus tendon may receive more direct exposure compared to the supraspinatus with this technique.However, the close proximity of the infra-and supra-supinatus tendons within the rotator cuff should ensure that healing of the supraspinatus tendon is enhanced by growth factors secreted from the adjacent infraspinatus tendon after in vivo transduction by AAV.
Although prior rotator cuff research has predominantly focused on the supraspinatus, some studies suggest the infraspinatus may play a more significant role in rotator cuff rupture (Jernheden and Szaro, 2022;Kato et al., 2012;Williams et al., 2023).For example, Mochizuki et al. (2008) showed tears involving the anterior greater tuberosity have substantial infraspinatus involvement, rather than just supraspinatus damage.Subsequently, Kato et al. (2012) also found a close relationship between the transverse infraspinatus and supraspinatus anatomically and functionally.www.ecmjournal.orgWhile tendon injuries are not lifethreatening, they can still have a major impact on quality of life.Rotator cuff injury that fails to heal properly often leads to chronic pain, reduced mobility, and decreased strength and function (Canosa-Carro et al., 2022).Previous studies have demonstrated the potential of various therapeutics biologics, such as TGF-β1, plateletderived growth factor subunit B (PDGF-B), GDF-5, e (BMP)-12, and BMP-14 utilizing gene delivery methods to enhance tendon repair (Bolt et al., 2007;Docheva et al., 2015;Hildebrand et al., 2004;Jin et al., 2022;Lou et al., 2001).Antagonists of inflammatory cytokines, such as interleukin (IL)-1, IL-2, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α), might also be beneficial to modulate the inflammatory response and prevent adhesion formation.This is particularly relevant as these cytokines play a role in the pathophysiological mechanism of subacromial inflammation (Sachinis et al., 2022).AAV2.5 is thus of considerable interest as a vector for delivering genes to regenerate injured tendons; which genes to transfer is a matter for future research.

Conclusions
Our comprehensive analysis of AAV serotypes in rat and human supraspinatus tenocytes identifies AAV2.5 as a highly efficient vector for gene transfer to tenocytes.This vector is able to transduce infraspinatus cells in situ following in vivo delivery into the subacromial space of the rat shoulder, delivering genes to both the main body of this tendon as well as the paratenon.Given the important role of the paratenon in tendon healing, the latter capability may be of high significance.In vivo transgene expression in the rat shoulder persists for 7 days, a period of time that may be well suited to initiating a robust regenerative response.Future research is needed to determine which transgenes provide the greatest regenerative response.
Polyethylenimine (PEI) was used to transfect 293T cells with the following 3 plasmids: helper www

Fig. 4 .
Fig. 4. In vivo expression of luciferase following injection of AAV2.5-Luciferase into subacromial space of the rat shoulder.(A) IVIS images of rats at various times following injection of AAV2.5-