Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10–16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%–72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%–12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.


Figure S1 .
Figure S1.The hydrodynamic diameter (d.nm), polydispersity index (PDI), and Zeta Potential (mV) of i) unloaded, ii) RNP-loaded, and iii) mRNA-loaded LNPs determined by dynamic light scattering.Data are depicted as the mean value of three independent measurements ± standard deviation (SD).DOPE-LNP encapsulation efficiencies (EE%) for Cas9 RNP and mRNA following bench-top mixing and microfluidic mixing (preloaded) as determined by iv) Ribogreen ® assay.

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Figure S2.A) Scheme summarizing the principle of a PrimeTime qPCR assay.B) Validation of the PrimeTime qPCR setup to detect indel formation.Assay validation was done by the addition of pre-synthesized genomic blocks to control genomic DNA samples of unedited samples.

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Figure S3.A) Frequency of indel % (normalized to wild-type (WT) cells) in the model gene HPRT after transfection of primary human keratinocytes (KCs) with 0%, 20%, or 40% DOTAP-containing LNP loaded with mRNA (N/P 6) or RNP (L/R 500).Data are presented as the mean of four biological replicates ± SD.B) Cell viability of KCs 48 h after exposure to DOTAP-containing LNP loaded with mRNA or RNP, respectively.Data are presented as the mean of three biological replicates ± SD.

Figure S4 .
Figure S4.Live dead staining of untreated human primary keratinocytes.

Figure S6 .
Figure S6.Skin cross-section of excised human skin after laser-induced microablation confirming the generation of the micropores.

Figure S7 :
Figure S7: Distribution of DiI-LNP in 3D skin models after microneedle (A-B) and laser (C-D) treatment.DiI-LNPs were added to skin models and incubated for 24 hours, after which skin models were frozen, cryo-sectioned, and imaged with a Keyence BZ-X810.E & F show cross-sections of untreated skin models.

Figure S8 .
Figure S8.Top: Representative histological hematoxylin & eosin staining of bioengineered 3D skin models after pre-treatment with solid microneedles showing the opening that is restricted to the epidermis.Bottom: Representative images of the 400 µm solid microneedle array that was used for the skin pre-treatment.

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Figure S9.A) Frequency of indel % (normalized to wild-type (WT)) in bioengineered 3D skin models (left) and freshly excised human skin (right) after pre-treatment with microneedles.n=3.B) Frequency of indel % (normalized to wild-type (WT)) in freshly excised human skin and bioengineered 3D skin models after transfection with Cas9 mRNA or RNP encapsulated in RNAiMax.

Figure S10 .
Figure S10.Release of IL6 and IL8 as exemplary makers for skin irritation from freshly excised human skin after pre-treatment with either laser microablation yielding pore formation in different skin depths or 400 µm microneedles (MN) prior the application of DOPE-based LNPs or LNP H.

Figure S11 .
Figure S11.rhAMP-Sequencing results of primary human keratinocytes after treatment with Cas9 mRNA-loaded LNP H.This table shows the relevant on-target and eight predicted off-target sites and corresponding sequences, the number of reads per site and the indel frequencies in percentage.

Figure S12 .
Figure S12.Visualization of the distribution of the most frequently identified alleles around the cleavage site for the sgRNA AATTATGGGGATTACTAGGA in donor 2. Nucleotides are indicated by unique colors (A = green; C = red; G = yellow; T = purple).Substitutions are shown in bold font.Red rectangles highlight inserted sequences.Horizontal dashed lines indicate deleted sequences.The vertical dashed line indicates the predicted cleavage site.

Figure S13 .
Figure S13.ARCI patient-derived cells were characterized by Sanger sequencing and Western blot clearly showing the mutation at c.877-2 A>G splice site.This mutation causes the expression of a truncated transglutaminase 1 protein which was confirmed by Western blot showing bands at 50 kDa while the full-length protein has a molecular weight of 92 kDa.