Preclinical Evaluation of a Modified Herpes Simplex Virus Type 1 Vector Encoding Human TGM1 for the Treatment of Autosomal Recessive Congenital Ichthyosis

Autosomal recessive congenital ichthyosis (ARCI) is a diverse group of cornification diseases associated with severe clinical complications and decreased quality life. Germline mutations in the TGM1 gene, which encodes enzyme TGM1, are predominant cause ARCI. These trigger abnormal epidermal differentiation impaired cutaneous barrier function observed patients Unfortunately, current ARCI therapies focus solely on symptomatic relief. Thus, there significant unmet need for therapeutic strategies aimed at correcting deficiency underlying In this study, we investigated ability KB105, gene therapy vector encoding full-length human to deliver functional keratinocytes. vitro, KB105 efficiently infected TGM1-deficient keratinocytes, produced protein, rescued transglutaminase function. vivo studies demonstrated that both single repeated topical administration induced protein expression target layer without triggering fibrosis, necrosis, or acute inflammation. Toxicity biodistribution assessments repeat dosing indicated was well-tolerated restricted dose site. Overall, our results demonstrate rescuing through application represents promising strategy safely noninvasively treating debilitating disease. heterogeneous rare affecting approximately 1:200,000 persons (Orphanet, 2020OrphanetPrevalence diseases: bibliographic data, Orphanet report series, collection, number 1: listed alphabetical order.https://www.orpha.net/orphacom/cahiers/docs/GB/Prevalence_of_rare_diseases_by_alphabetical_list.pdfDate: 2020Google Scholar). Whereas pathogenic least 12 genes have been identified ARCI, germline affect >55% those United States (Rodríguez-Pazos et al., 2013Rodríguez-Pazos L. Ginarte M. Vega A. Toribio J. ichthyosis.Actas Dermosifiliogr. 2013; 104: 270-284Crossref PubMed Scopus (27) Google Scholar) up 84% cases Norway (Farasat 2009Farasat S. Wei M.H. Herman Liewehr D.J. Steinberg S.M. Bale S.J. al.Novel transglutaminase-1 genotype-phenotype investigations 104 autosomal USA.J Med Genet. 2009; 46: 103-111Crossref (68) Scholar; Pigg 1998Pigg Gedde-Dahl Jr., T. Cox D. Hausser I. Anton-Lamprecht Dahl N. Strong founder effect 1 mutation lamellar ichthyosiform erythroderma from Norway.Eur J Hum 1998; 6: 589-596Crossref (44) an intracellular plays critical role formation cornified envelope (CE), structure acts as mechanical against transepidermal water loss entry infectious agents. mainly localized within stratum granulosum, corneum, hair follicles, where it catalyzes covalent cross-linking different CE proteins (Candi 2005Candi E. Schmidt R. Melino G. The envelope: model cell death skin.Nat Rev Mol Cell Biol. 2005; 328-340Crossref (1202) Loss activity compromises leads function, initiating default compensatory pathway hyperproliferation resulting presentation and/or generalized scaling (Yang 2016Yang C.S. Pomerantz H. Mannava K.A. Corwin Weinstock M.A. Fleckman P. al.Comparing histopathology X-linked mutation: National Registry Ichthyosis Related Skin Disorders.J Am Acad Dermatol. 2016; 74: 1008-1010.e2Abstract Full Text PDF (2) Disruptive lead chronic often devastating defective (Herman 2009Herman M.L. Farasat Steinbach P.J. Toure O. al.Transglutaminase-1 ichthyosis: summary (including 23 novel) modeling TGase-1.Hum Mutat. 30: 537-547Crossref (65) Individuals TGM1-associated typically born encased tight, shiny collodion membrane, sheds during first weeks Neonatal occur 45% cases, leading infant mortality rate ∼11% (Chung 2011Chung Pittenger Tobin Chung Desai Expedient treatment baby.Case Rep Dermatol Med. 2011; 2011: 803782PubMed After shedding plate-like scales develop skin usually entire integument. Other signs symptoms include predisposition heat intolerance, eclabium ectropion, loss, palmoplantar hyperkeratosis, nail abnormalities, conductive hearing respiratory problems. Quality life negatively impacted young age, life-long social stigmatization, shyness, 4 hours care daily (Gånemo 2003Gånemo Virtanen Kukk Raudsepp Rossman-Ringdahl al.Autosomal Sweden Estonia: clinical, genetic ultrastructural findings eighty-three patients.Acta Derm Venereol. 2003; 83: 24-30Crossref (35) options provide only For involvement, retinoid prescribed; however, retinoids fail restore carry potential complications. There substantial molecularly corrects investigate TGM1-modifying effects replication-defective herpes simplex virus type TGM1. infects relevant cells rescues vivo, yields toxicity when applied topically immunocompetent animals. epidermis, readministration sustains high DNA RNA levels while simultaneously boosting levels. Finally, evaluation weekly redosing indicates remains limited Taken together, these preclinical proof-of-concept safety strong support recurrent use safe effective Purified evaluated transduction efficiency effector two-dimensional cell-based assays. assays employed immortalized keratinocytes (KCs) harvested patient homozygous c.877-2A>G splice-site mutation, most commonly reported humans Cells were multiplicities infections (MOIs) ranging 0.3 3.0 48 hours, analyzed by qPCR, quantitative real-time RT-PCR, western blot, immunofluorescence (IF). Negative controls included uninfected (mock) mCherry-expressing (mCherry). genomes transcript detected ARCI-derived KCs MOI low showed dose-dependent increase (Figure 1a) 1b) Increased blot immunofluorescent analysis relative mock-infected 1c d). No detectable endogenous KCs, confirming isolated harboring natural deficiency.Figure 1In vitro assessment primary grown low-calcium culture medium. (a) Dose-dependent detection copies increasing MOIs assessed qPCR. Data presented average two replicates ± SEM. (b) transcripts QRT-PCR. (c) KB105-mediated blot. (d) Representative IF images infection KCs. (e) KB105-dependent enzymatic (f) analysis. (g) cells. experiments, HSV-mCherry‒infected (mCherry) used negative controls; NPKs positive control. DAPI staining visualize nuclei. GAPDH loading analyses, quantification fluorescence intensities provided each condition. Bar = 130 μm. ichthyosis; HSV, virus; IF, immunofluorescence; KC, keratinocyte; MOI, multiplicity infection; NPK, normal KC; QRT-PCR, RT-PCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT) functionality KB105-expressed next examined determining whether exogenous catalyzed between glutamine lysine residues, essential TGM1-mediated assembly CE. Protein using situ TGM1-specific peptide assay employing biotinylated mimics substrate. conjugation peptides visualized incubating treated fluorescently labeled streptavidin. A KB105-infected surpassing 1e). Uninfected no activity. similar trend subsequent restoration calcium medium stimulate (Supplementary S1a b). transduce more clinically type, is, examined. Restoration 1f). As expected, control Supporting data revealed 1.0 1g). rescue also after growth high-calcium S2). impact morphology environments S3a). Mild cytotoxic dosages cells, may account 3 analyses S3b). Because deletion neonatally lethal mice (Matsuki 1998Matsuki Yamashita F. Ishida-Yamamoto Yamada K. Kinoshita C. Fushiki al.Defective corneum early neonatal lacking (keratinocyte transglutaminase).Proc Natl Sci USA. 95: 1044-1049Crossref (240) Scholar), mouse lesion not practicable evaluations. such, pharmacology conducted BALB/c mice. This approach allowed determination vector’s properly concurrently monitoring fully intact immune system. First, mechanically chemically disrupted dorsal Sequential tape stripping wiping surface acetone techniques disruption (Rissmann 2009Rissmann Oudshoorn Hennink W.E. Ponec Bouwstra J.A. acetone: observations hairless model.Arch Res. 301: 609-613Crossref (31) formulated methylcellulose gel carrier administered regions prepared mouse. biopsies processed histological examination samples evaluate KB105-induced physiological changes, indicate concerns vivo. obvious inflammation any KB105-treated compared vehicle 2a). Post-killing qPCR effectively transduced acetone-treated– strip–permeabilized 2b), expressed 2c). found induce transcription, differences low- high-dose S4).Figure 2In multiple routes delivery H&E-stained tape-stripped permeabilized either (low dose) (vehicle). permeabilization (vehicle), condition QRT-PCR tissue (two tested per sample) SEM; condition, four loricrin, integrin alpha-6 localization 50 Exogenous IF. Human epidermis 2d). Paralleling RT-PCR results, qualitative high- versus low-dose samples. Samples costained loricrin (a substrate TGM1) marker basal epidermis) determine exogenously originating correctly functionally active. Mouse colocalized all samples, whereas superficial than alpha-6. Together, successfully targeted layer. short-term pharmacokinetics study S5). Vector remained relatively stable over course 48-hour numbers 8- timepoints. 2 steadily increased time, peaking 24 before declining (while remaining detectable) hours. Similar transgene kinetics level, We feasibility applications intervals (days vs. days 12). Briefly, stripped (or control) day 1. tissues cohort act (KB105) (vehicle) controls. Additional cohorts re–tape retreated 12, subsequently 5 14, respectively. Histological reorganization, including inflammation, 3a). High genome copy KB105; comparable 3b). receiving second 3c).Figure 3In taken timepoints Detection analysis, six colocalization qualitatively measured. Epidermal loricrin. Significant well proper once twice 3d). some variability cohorts, gross KB105. Good Laboratory Practice repeat-dose male female (Table 1). Mice dosed week (group 2) 1) stripping. Six sex one necropsied 3. animals 1, 8, 15, 22, 29 30. All surviving 29, then subjected 33-day recovery phase necropsy.Table 1Design GLP Repeat-Dose Biodistribution StudyGroup No.No. Animals1Mice designated interim necropsy (six group) euthanized phase. terminal 30 underwent completion 34 (day 63 phase).Dose Level (PFU/Application)MaleFemale1(Vehicle control)2Group excipient only.181802(KB105)18181.07 × 109Abbreviations; GLP, Practice; No., number; PFU, plaque-forming unit.1 phase).2 Group only. Open table new tab Abbreviations; unit. Assessment based mortality, observations, body weights, food consumption, dermal anatomic pathology. KB105-related weight consumption macroscopic findings, organ parameters noted. survived until their scheduled necropsy. Microscopic selected tissues, site, sternum bone, bone marrow, brain, epididymis, heart, kidneys, liver, lungs, axillary lymph node, inguinal ovaries, oviducts, prostate, spleen, testes, thymus, uterus cervix. epithelial hyperplasia, edema, killings. consistent repair abrasion considered related procedure. At killing, exhibited complete reversibility procedure‒related microscopic findings. adverse level be 1.07 109 unit/day. Blood biodistribution. Nearly blood collected three except dose-site root contamination occurred preparation specifically generally pronounced accumulation other Table S1). persistence minimal, low-to-negative obtained recovery-phase Reliable paramount feature required defect reproducibly efficiency, capably expression, restored otherwise Subsequent successful native substrate, signifying targeting granulosum. rapidly skin, genomes, transcripts, observable administration. its encoded treatment, irrespective length delay treatments. histology well-tolerated, even dosages. can repeatedly systemic exposure. transient prior proceeded trials States. However, several research groups explored methods address diseased phenotype virus-mediated approaches (Choate 1996Choate Medalie D.A. Morgan J.R. Khavari P.A. Corrective transfer disorder ichthyosis.Nat 1996; 2: 1263-1267Crossref (144) liposomally delivered purified recombinant (Aufenvenne 2013Aufenvenne Larcher Duarte B. Oji V. Nikolenko al.Topical enzyme-replacement restores transg