QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive (RDEB) or dominant (DDEB) and associated with high wound burden. Perpetual cycles of wounding healing drive fibrosis DDEB RDEB, well formation tumor-permissive microenvironment. Prolonging wound-free episodes improving quality would therefore confer substantial benefit for individuals DEB. The collagenous domain VII encoded 82 in-frame exons, which makes splice-modulation therapies attractive Indeed, antisense oligonucleotide–based exon skipping has shown promise RDEB. However, suitability oligonucleotides treatment remains unexplored. Here, we developed QR-313, clinically applicable, potent oligonucleotide specifically targeting 73. We show feasibility topical delivery QR-313 carbomer-composed gel wounds to restore abundance human RDEB skin. Our data reveal that also shows direct 73 mutations. Thus, same topically applied therapeutic could used improve DDEB. genetic disorder, either recessively dominantly (DDEB). In normal skin, forms anchoring fibrils link dermis epidermal basement membrane (Keene et al., 1987Keene D.R. Sakai L.Y. Lunstrum G.P. Morris N.P. Burgeson R.E. Type an extended network fibrils.J Cell Biol. 1987; 104: 611-621Crossref PubMed Scopus (274) Google Scholar). Absence functional leads impaired fibril formation, causing fragility mucosal membranes. Apart from blistering, patients suffer erosions, esophageal strictures, progressive fibrosis, ultimately aggressive squamous cell carcinoma (Fine, 2010Fine J.D. Inherited bullosa.Orphanet J Rare Dis. 2010; 5: 12Crossref (134) Currently, patient care limited palliative measures (El Hachem 2014El M. Zambruno G. Bourdon-Lanoy E. Ciasulli A. Buisson C. Hadj-Rabia S. al.Multicentre consensus recommendations 2014; 9: 76Crossref (75) several curative approaches are being evaluated clinical trials, including autologous therapy grafts gene-corrected keratinocytes (KCs) (Eichstadt 2019Eichstadt Barriga Ponakala Teng Nguyen N.T. Siprashvili Z. al.Phase 1/2a trial dystrophic bullosa.JCI Insight. 2019; 4: e130554Crossref (32) Scholar; 2016Siprashvili Gorell E.S. Loutit K. Khuu P. Furukawa L.K. al.Safety outcomes following genetically corrected bullosa.JAMA. 2016; 316: 1808-1817Crossref (103) Scholar) injections fibroblasts (Lwin 2019Lwin S.M. Syed F. Di W.L. Kadiyirire T. Liu L. Guy early efficacy lentiviral fibroblast e126243Crossref (24) despite these positive advances, it clear complementary options urgently required (Bruckner-Tuderman, 2019Bruckner-Tuderman Newer modalities bullosa.Indian Dermatol Online J. 10: 244-250Crossref Although DEB-causing spread rather evenly over gene, there certain mutational hotspots, one. Mutations this cause (Varki 2007Varki R. Sadowski Uitto Pfendner Epidermolysis bullosa. II. type phenotype-genotype correlations subtypes.J Med Genet. 2007; 44: 181-192Crossref (179) RNA modulation splicing pre-mRNA employed omit specific exons mRNA. candidate exon-skipping majority its short individually removed without disturbing open reading frame (Bornert 2016Bornert O. Kühl Bremer van den Akker P.C. Pasmooij A.M. Nyström Analysis consequences targeted deletion reveals prospects therapy.Mol Ther. 24: 1302-1311Abstract Full Text PDF Exon use (AONs) been successfully 13, 70, 73, 80, 105 2016Bremer Bornert Gostynski Jonkman M.F. Aartsma-Rus al.Antisense oligonucleotide-mediated systemic approach bullosa.Mol Ther Nucleic Acids. e379Abstract (37) Goto 2006Goto Sawamura D. Nishie W. McMillan J.R. Akiyama al.Targeted single harboring premature termination codon mutation: implications potential correction selective patients.J Invest Dermatol. 2006; 126: 2614-2620Abstract (57) Turczynski 2016Turczynski Titeux Tonasso Décha Ishida-Yamamoto Hovnanian Targeted restores expression vivo model.J 136: 2387-2395Abstract (40) Thus far, work focused on little known about AONs DDEB, although represents around 40% cases 2016Fine Epidemiology based incidence prevalence estimates National Bullosa Registry.JAMA 152: 1231-1238Crossref Recent natural history studies suggested no even negative effects (Bremer 2019aBremer der Heijden E.H. Eichhorn D.S. Meijer Lemmink H.H. Scheffer H. al.Natural sets stage 18: 465-475Abstract (10) experimental assessment disease-relevant setting not performed. Specifically synthesis restored intradermal nonclinical grade equivalents composed compound heterozygous 73–mutated cells grafted onto mice (Turczynski Chronically injured inflamed extreme pain after injections. injectable drugs require repeated administrations unfavorable candidates. To allow translation AON number DEB, developing efficient therapeutically optimized After reconfirming lacking amino acids functional, designed manufacturability safe relevant concentrations was formulated carbomer-based hydrogel. Topical application epidermal-dermal cohesion, led distribution bed wounded equivalents, promoted subsequently tested KCs (DDEBKs), where showed evidence ex models. Collectively, provide rationale (NCT 03605069) evaluating people This study aimed develop applicable Toward end, most frequently mutated (Mecklenbeck 1999Mecklenbeck Hammami-Hauasli N. Höpfner B. Schumann Kramer Küster al.Clustering 73: mutation analysis bullosa.J 1999; 112: 398-400Abstract (27) Because evaluation therapeutics more setting, first mimic restoration production mutation-carrying biallelic null situation, expressed homotrimeric (?73 VII). Previous have indicated ?73 support previous observations, subtle changes stability functionality collagens pathological activity (Zou 2017Zou Y. Donkervoort Salo Foley A.R. Barnes Hu al.P4HA1 unique congenital disorder connective tissue involving tendon, bone, muscle eye.Hum Mol 2017; 26: 2207-2217Crossref (20) Scholar), performed careful biochemical wild-type (WT) were secreted at similar levels (Figure 1a). Correctly folded triple helices resistant trypsin proteolysis. Limited exposure vis-à-vis WT 30 °C higher P1 into thermostable helix 1a b) Changes may alter binding interaction partners Solid-phase assays immobilized IV laminin-332 bound both proteins affinity VII, whereas denatured did 1c). Additional retained came ability (RDEBF) adhesion KC (RDEBK) migration (Supplementary Figure S1), surrogate marker Woodley 2008Woodley D.T. Hou Martin Li Chen Characterization molecular mechanisms underlying using site-directed mutagenesis.J Biol Chem. 2008; 283: 17838-17845Abstract (26) obtain functionality, 10 ?g purified injected twice intradermally model mice. stained antibodies days injection (Kühl 2015Kühl Mezger Hausser I. Handgretinger Bruckner-Tuderman High local MSCs integrity facilitate 2015; 23: 1368-1379Abstract (46) localized dermal-epidermal junction (DEJ) 1d). Transmission electron microscopy analyses revealed clearly visible fibrils. partially rescued width lamina densa 1e f), reduced hypomorphic (Nyström 2013Nyström Velati Mittapalli V.R. Fritsch Kern J.S. Collagen plays dual role healing.J Clin Invest. 2013; 123: 3498-3509Crossref (115) Together, our disclose any discernable differences level between covers 201 base pairs, making largest domain–encoding flexibility design exon. flanked two introns (86 96 respectively) followed 74 (36 pairs). arrangement challenge design, coskipping when observed before Partial coremoval create additional polypeptide should thus avoided. AONs, silico analyses, taking account optimal placement, length, sequence features S2 Supplementary Table S1). yielded candidates, synthesized screened absence coskipping. skip efficiency favorable chemical composition, selected further development 2a). Dose-dependent (normal 1 2) RDEBFs (RDEBF-muE73-1 RDEBF-muE73-2) transfection 0 200 nM slightly than control (half maximal effective concentration, 9.23 ± 1.7 12.5 1.4 nM, reached plateau donors Next, response assessed homozygous (RDEBK-muE73-3). Correct confirmed PCR, sequencing 2b S3a). Immunofluorescence staining QR-313–mediated RDEBK-muE73-2 2c). Further, western blot AON-treated RDEBK-muE73-3 supernatants corresponding (27% 30% 60 Testing one donor (RDEBK-muE73-1) independency S3b). Altogether, supported good AON-based therapy. assess functionally cohesion tractable model, (HSEs). HSEs together completely VII–deficient (RDEBF-1) (Table 1) fibroblasts. At start coculture every other day until end experiment, 100 added dropwise RDEBK-muE73-2. Analyses three weeks significantly DEJ 3a). separation 3b), showing promotes strengthening integrity.Table 1Characteristics Cells Used StudyCodeDonor TypeMutationsRemarksNHK-1, 2, 3, 4WT—Immortalized KCsNHF-1, 4WT—Primary fibroblastsRDEBK-muE73-1RDEB, 73c.425A>G, c.6022C>TPrimary immortalized KCsRDEBK- muE73-2RDEB, 73c.6081 delC, c.6081 delCImmortalized KCsRDEBK-3RDEB severe generalized, deficientc.425A>G, c.425A>GImmortalized KCsRDEBF- muE73-1RDEB, 73c.6176A>G, c.6501G>APrimary fibroblastsRDEBF- fibroblastsRDEBF-1RDEB c.425A>GPrimary fibroblastsDDEBK-muE73-1DDEB, 73c.6118C>AImmortalized KCsDDEBK-muE73-2DDEB, 73c. 6127 G>AImmortalized KCsAbbreviations: bullosa; DDEBK, keratinocyte; KC, NHF, fibroblast; NHK, RDEBF, RDEBK, WT, wild type. Open table new tab Abbreviations: A prerequisite sustainable drug frequent Delivery through epidermis challenge. benefits obtained transepidermal because burden, edges epithelium ideal assembles consequently constitutes area increased enable rapid clinics, approved formulations hypromellose- gels. Given close similarities barriers porcine transdermal (Abd 2016Abd Yousef S.A. Pastore M.N. Telaprolu Mohammed Y.H. Namjoshi al.Skin models testing drugs.Clin Pharmacol. 8: 163-176PubMed Based best diffusion dermal Cy-5–conjugated (not shown), hydrogel unwounded concentration 50 mg/cm2 overt dermis, FISH 3c, upper panels). contrast, equal amount resulted uptake tongues, bed, adjacent lower These main VII–producing during regeneration applicability formulation. HSEs, 1). blister wounds, partly stripped center HSE. Directly wounding, formulation placebo times week harvesting. primarily 3d). different regions HSE dissected analyzed Droplet Digital PCR treatment. Skipping strictly increase time treatment, intact 3e). contribute minimally deposition (Marinkovich 1993Marinkovich M.P. Keene Rimberg C.S. Cellular origin membrane.Dev Dyn. 1993; 197: 255-267Crossref (205) S4). fibroblasts, given low wound-adjacent expected. Nevertheless, immunostained sections de novo S5a). Immunogold-labeled NC1 NC2 domain-specific (NP185 LH24) transmission some positivity QR-313–treated but contrast proven S5b shown). As previously mentioned, disease-causing result hold great generally milder form Treatment carry allele allele, raises concerns and/or dominant, disease-aggravating shortened heterotrimers considered initially Despite challenges, efficiently induce correct carrying missense 4a S6). There notable difference potency 11.5 1.6 respectively). DDEBK S7). hallmark helical thermostability digestion resistance DDEBK-produced proteolysis 4b c). if improved lead improvement, vitro markers. On culture, plastic bind laminin-332; however, crucial DDEBKs 4d e). No seen indicating effect detachment 4f g). tendency extracellular S8). appeared broad fuzzy, collectively indicate attachment due QR-313–evoked improvement functionality. Finally, elucidate course action protein HEK-293 singly dually transfected WT-COL7A1 cDNA constructs His- FLAG-tags FLAG-tagged ?73-COL7A1 construct 5a). ELISA FLAG-trapped that, ?1 chain polypeptides interacted, occurred 5b Single sequential purification observations 5d). Furthermore, cellular stress, consequence misfolded molecules, occur S9). Taken together, safety prospective By bolstering applications able significantly. showe