Adeno-associated virus (AAV)-based gene therapy has been shown to be safe and effective in numerous animal models and clinical trials for various ophthalmic diseases. Stargardt disease (STGD1; MIM #248200) is the most common autosomal recessive macular dystrophy disease, and the most common form is caused by mutations in the ABCA4 gene, a gene with 6.8 kb coding sequence. Split intein approaches increase the capacity of dual AAV gene therapy, but at the cost of reduced protein expression, which may be insufficient to achieve a therapeutic effect. In this study, we designed various dual split intein ABCA4 vectors and showed that the efficiency of expression of full-length ABCA4 protein is dependent on combinations of types and split sites of the intein system. The most efficient vectors were identified through in vitro screening, and a novel dual AAV8-ABCA4 vector was constructed and subsequently proven to express full-length ABCA4 protein at a high level, reducing bisretinoid formation and correcting the visual function of ABCA4-knockout mice. Furthermore, we evaluated therapeutic effects of different dosages by subretinal injection in mice model. Both therapeutic effects and safety were guaranteed under the treatment of 1.00 × 109 GC/eye. These results support the optimized dual AAV8-ABCA4 approach in future clinical translation for treatment of Stargardt disease.
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References
1.
WaliaS, FishmanGA. Natural history of phenotypic changes in Stargardt macular dystrophy. Ophthalmic Genet, 2009; 30(2):63–68.
2.
AndersonKL, BairdL, LewisRA, et al.A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p. Am J Hum Genet, 1995; 57(6):1351–1363.
3.
StoneEM, AndorfJL, WhitmoreSS, et al.Clinically focused molecular investigation of 1000 consecutive families with inherited retinal disease. Ophthalmology, 2017; 124(9):1314–1331.
4.
RaduRA, MataNL, BaglaA, TravisGH. Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration. Proc Natl Acad Sci U S A, 2004; 101(16):5928–5933.
5.
MoldayRS, ZhangK. Defective lipid transport and biosynthesis in recessive and dominant Stargardt macular degeneration. Prog Lipid Res, 2010; 49(4):476–492.
6.
XieYL, WangJ, HeY. The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo. J Integr Med, 2023; 21(1):106–115.
7.
FischerMD, MichalakisS, WilhelmB, et al.Safety and vision outcomes of subretinal gene therapy targeting cone photoreceptors in achromatopsia: A nonrandomized controlled trial. JAMA Ophthalmol, 2020; 138(6):643–651.
8.
MishraA, VijayasarathyC, CukrasCA, et al.Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial. Mol Ther, 2021; 29(6):2030–2040.
9.
Fuller-CarterPI, BasiriH, HarveyAR, et al.Focused update on AAV-based gene therapy clinical trials for inherited retinal degeneration. BioDrugs, 2020; 34(6):763–781.
10.
Voretigene neparvovec-rzyl (Luxturna) for inherited retinal dystrophy. Med Lett Drugs Ther, 2018;60(1543):53–55.
11.
CrabtreeE, UribeK, SmithSM, et al.Inhibition of experimental autoimmune uveitis by intravitreal AAV-Equine-IL10 gene therapy. PLoS One, 2022; 17(8):e0270972.
12.
KapranovP, ChenL, DederichD, et al.Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing. Hum Gene Ther, 2012; 23(1):46–55.
13.
HirschML, LiC, BellonI, et al.Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway. Mol Ther, 2013; 21(12):2205–2216.
14.
McClementsME, IssaCP, BlouinV, et al.A fragmented adeno-associated viral dual vector strategy for treatment of diseases caused by mutations in large genes leads to expression of hybrid transcripts. J Genet Syndr Gene Ther, 2016; 7(5):311.
15.
TrapaniI, ColellaP, SommellaA, et al.Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med, 2014; 6(2):194–211.
16.
McClementsME, BarnardAR, SinghMS, et al.An AAV dual vector strategy ameliorates the Stargardt phenotype in adult Abca4-/- mice. Hum Gene Ther, 2019; 30(5):590–600.
17.
DykaFM, BoyeS, ChiodoVA, et al.Dual adeno-associated virus vectors result in efficient in vitro and in vivo expression of an oversized gene MYO7A. Hum Gene Ther Methods, 2014; 25(2):166–177.
18.
TornabeneP, TrapaniI, MinopoliR, et al.Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Sci Transl Med, 2019; 11:492.
19.
ColellaP, TrapaniI, CesiG, et al.Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors. Gene Ther, 2014; 21(4):450–456.
20.
NovikovaO, TopilinaN, BelfortM. Enigmatic distribution, evolution, and function of inteins. J Biol Chem, 2014; 289(21):14490–14497.
21.
MillsKV, JohnsonMA, PerlerFB. Protein splicing: How inteins escape from precursor proteins. J Biol Chem, 2014, 289(21):14498–14505.
22.
LiY. Split-inteins and their bioapplications. Biotechnol Lett, 2015; 37(11):2121–2137.
23.
LockM, AlviraM, VandenbergheLH. Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum Gene Ther, 2010; 21(10):1259–1271.
24.
Charbel IssaP, SinghMS, LipinskiDM, et al.Optimization of in vivo confocal autofluorescence imaging of the ocular fundus in mice and its application to models of human retinal degeneration. Invest Ophthalmol Vis Sci, 2012; 53(2):1066–1075.
25.
WuY, FishkinN, PandeA, et al.Novel lipofuscin bisretinoids prominent in human retina and in a model of recessive Stargardt disease. J Biol Chem, 2009; 284(30):20155–20166.
26.
Van't VeerA, BechtholtA, OnvaniS. Ablation of kappa-opioid receptors from brain dopamine neurons has anxiolytic-like effects and enhances cocaine-induced plasticity. Neuropsychopharmacology, 2013; 38(8):1585–1597.
27.
FoxMW. The visual cliff test for the study of visual depth perception in the mouse. Anim Behav, 1965; 13(2):232–233.
28.
MataNL, WengJ, TravisGH. Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration. Proc Natl Acad Sci U S A, 2000; 97(13):7154–7159.
29.
RussellS, BennettJ, WellmanJA. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: A randomised, controlled, open-label, phase 3 trial. Lancet, 2017; 390(10097):849–860.
30.
PatelU, BoucherM, de LéséleucL, et al.Voretigene Neparvovec: An Emerging Gene Therapy for the Treatment of Inherited Blindness. In: CADTH Issues in Emerging Health Technologies. Canadian Agency for Drugs and Technologies in Health: Ottawa, ON;, 2018; pp. :1–11.
31.
ZhengQ, WangT, ZhuX. Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo. Mol Ther Methods Clin Dev, 2021; 22:293–303.
32.
GrishaninR, VuillemenotB, SharmaP. Preclinical evaluation of ADVM-022, a novel gene therapy approach to treating wet age-related macular degeneration. Mol Ther, 2019; 27(1):118–129.
33.
GelfmanCM, GrishaninR, BenderKO. Comprehensive preclinical assessment of ADVM-022, an intravitreal anti-VEGF gene therapy for the treatment of neovascular AMD and diabetic macular edema. J Ocul Pharmacol Ther, 2021; 37(3):181–190.
34.
ZoborD, WernerA, StanzialF. The clinical phenotype of CNGA3-related achromatopsia: Pretreatment characterization in preparation of a gene replacement therapy trial. Invest Ophthalmol Vis Sci, 2017; 58(2):821–832.
35.
MacLarenRE, GroppeM, BarnardAR. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial. Lancet, 2014; 383(9923):1129–1137.
36.
XueK, JollyJK, BarnardAR. Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia. Nat Med, 2018; 24(10):1507–1512.
37.
MaddalenaA, TornabeneP, TiberiP. Triple vectors expand AAV transfer capacity in the retina. Mol Ther, 2018; 26(2):524–541.
38.
DykaFM, MoldayLL, ChiodoVA, et al.Dual ABCA4-AAV vector treatment reduces pathogenic retinal A2E accumulation in a mouse model of autosomal recessive Stargardt disease. Hum Gene Ther, 2019; 30(11):1361–1370.
39.
TornabeneP, TrapaniI, MinopoliR, et al.Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Sci Transl Med, 2019; 11(492):eaav4523.
40.
ChenYX, ZhiSY, LiuWL, et al.Development of highly efficient Dual-AAV split adenosine base editor for in vivo gene therapy. Small Methods, 2020; 4:2000309.
41.
ZetscheB, VolzSE, ZhangF. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol, 2015; 33(2):139–142.
42.
ChewWL, TabebordbarM, ChengJK. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods, 2016; 13(10):868–874.
43.
TruongDJ, KühnerK, KühnR. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res, 2015; 43(13):6450–6458.
44.
ArankoAS, WlodawerA, IwaïH. Nature's recipe for splitting inteins. Protein Eng Des Sel, 2014; 27(8):263–271.
45.
TrapaniI, TorielloE, de SimoneS, et al.Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease. Hum Mol Genet, 2015; 24(23):6811–6825.
46.
XiongW, WuD, XueY. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc Natl Acad Sci U S A, 2019; 116(12):5785–5794.
47.
Le MeurG, LebranchuP, BillaudF. Safety and long-term efficacy of AAV4 gene therapy in patients with RPE65 Leber congenital amaurosis. Mol Ther, 2018; 26(1):256–268.
48.
BainbridgeJW, MeharMS, SundaramV. Long-term effect of gene therapy on Leber's congenital amaurosis. N Engl J Med, 2015; 372(20):1887–1897.
49.
MaguireAM, SimonelliF, PierceEA, et al.Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med, 2008; 358(21):2240–2248.
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