Molecular Therapy

Achieving efficient and balanced transgene expression in both rods and cones remains a major challenge in retinal gene therapy. Current promoters either lack specificity or fail to provide sufficient cellular coverage and expression level. To address this limitation, we developed and evaluated two fusion promoters, Pikali and Nocchu, by combining PR1.7, a cone-specific promoter and GRK1, a promoter most active in rods. Here, we show that Pikali and Nocchu outperform their parental promoters, driving broader and more balanced GFP expression in rods and cones of human iPSC-derived retinal organoids. These constructs achieved transduction in 30% to 45% of photoreceptors, with higher expression levels than GRK1 and broader cellular coverage than PR1.7. Our findings establish Pikali and Nocchu as excellent candidates for retinal gene therapy, overcoming the limitations of existing promoters. By combining specificity, efficiency, and extensive photoreceptor targeting, these fusion constructs represent a novel and promising strategy for next-generation gene therapy vectors, addressing inherited retinal dystrophies and advancing clinical translation.

Adeno-associated virus (AAV) gene therapy holds immense promise for treating muscular dystrophies, yet its efficacy and safety are constrained by the suboptimal tissue tropism of natural serotypes. Here, we employed the REACH platform, which combines rational design and directed evolution, to engineer muscle targeting vectors. Systemic administration in non-human primates (NHPs) revealed that lead candidate M1 mediates a >10-fold increase in skeletal muscle transduction compared to the AAV9 and 2-3 fold higher than MyoAAV, while concurrently achieving a remarkable 183-fold reduction in liver distribution. Furthermore, M1 exhibited significant de-targeting from key off-target tissues, including dorsal root ganglia (11 fold), lung (27 fold), spleen (2 fold), and kidney (2 fold). These findings demonstrate that the REACH platform can generate AAV capsids with simultaneously enhanced muscle tropism and favorable safety profiles, addressing a critical bottleneck in muscle-directed gene therapy.

We report the discovery of novel adeno-associated virus (AAV) capsid variants engineered for superior intravitreal (IVT) gene delivery to the primate retina. Utilizing the REACH platform, we constructed a diverse AAV variant library and employed a multi-stage screening strategy involving in vitro selection on human retinal pigment cells followed by direct in vivo screening in non-human primates (NHPs). Following IVT administration in NHPS of a barcoded variant pool, next-generation sequencing analysis of retinal tissues identified lead candidates (e.g., E52, E54, and E57) that achieved transduction levels in the neural retina and RPE 5-10 fold higher than the benchmark R100. Concurrently, these high-potency variants exhibited an exceptional ocular confinement profile, with minimal to undetectable vector genome distribution in systemic organs. This combination of markedly enhanced retinal transduction and stringent local tropism establishes these engineered capsids as promising next-generation vectors for the treatment of inherited and acquired retinal diseases via a minimally invasive IVT route.

Gene therapies have demonstrated transformative potential for a range of genetic disorders, including immunodeficiencies, hematopoietic conditions, and neuromuscular diseases. However, the application of these approaches to cystic fibrosis (CF) and other airway diseases remains constrained by the challenge of efficient gene delivery to target epithelial cells. Adeno-associated virus (AAV) vectors are widely used for in vivo gene delivery due to their favorable safety profile and capacity for long-term transgene expression in non-dividing cells. Nonetheless, current AAV capsids require high doses to achieve therapeutic efficacy in the airways, raising safety concerns. Here we report the development of novel AAV capsid variants with markedly enhanced transduction efficiency of airway epithelial cells. Using unbiased peptide-modified AAV libraries and round-over-round screening in well-differentiated primary cultures of human airway epithelia (HAE), we identified 20 novel capsids that efficiently transduced cells at doses 10- to 100-fold lower than those required by existing vectors (termed AAV-AE). These variants demonstrated high transgene expression in HAE, primary human basal cells, tracheal explants from nonhuman primates, and murine airways in vivo. These optimized AAV capsids represent a significant advancement in pulmonary gene therapy, offering a versatile platform for the delivery of gene addition and editing reagents to treat CF and other respiratory diseases.

Adeno-associated virus (AAV)-based gene therapy has made steady progress towards efficient delivery to numerous target cell populations, yet the virus's 5 kb packaging limit remains a challenge for effective and in some cases cell-selective cargo expression. Here, we introduce Expression-Linked Promoter Selection (ELiPS), a high-throughput platform for generating and functionally screening >106 engineered, short promoter variants using an AAV expression platform. ELiPS relies on a Golden Gate cloning method to build random oligomers of selected transcription factor binding sites (TFBSs) upstream of a minimal promoter, GFP, and a unique 3' barcode. As a proof of concept, to engineer short (~250 bp), synthetic, ubiquitous promoters, we applied ELiPS to build two libraries composed of TFBSs for ubiquitously expressed transcription factors (TFs) and screened them via AAV-mediated transduction in vitro. This strategy identified promoters with expression surpassing human cytomegalovirus (CMV) and CAG in vitro, and one variant was capable of driving therapeutic expression of B-domain-deleted Factor VIII (BDDFVIII) in vivo at levels comparable to a liver-specific promoter benchmark. ELiPS thus establishes a scalable framework for promoter discovery, enabling the design of compact, ubiquitous or cell-selective expression cassettes that enable further precision and efficacy in AAV-based gene therapies.

To facilitate the translation of NF1 exon 17 skipping as a mutation-specific therapy for Neurofibromatosis type 1 into in vivo testing, we have continued to develop more efficient antisense oligonucleotides (ASOs), humanized mouse models, and explored multiple delivery platforms including an adeno-associated virus (AAV)-U7-SnRNA vector approach. We evaluated both biodistribution and exon skipping efficacy of a U7-SnRNA targeting NF1 exon 17 with an SFFV-driven cassette containing T2A-linked Luciferase (Luc) and eGFP packaged in AAV-9, AAV-F and AAV-B1 capsids. We show that AAV-F is superior to AAV-9 and AAV-B1 for mouse brain delivery based on DNA transduction, GFP expression, and luciferase activity, but AAV-B1 delivers 2-4 fold more to sciatic nerve (SCN). In terms of exon skipping, AAV-F appears to induce the most skipping in liver and optic nerve (ON), while AAV-B1 mediates highest skipping in the liver, SCN, and ON. The identification of AAV serotypes that allow efficient transduction and delivery of transgenes to the mouse CNS and PNS is impactful for preclinical research in murine models of other diseases. Furthermore, this is both the first report of NF1 exon skipping efficacy in vivo and the first successful application of an U7-SnRNA for the restoration of functional neurofibromin for NF1.

IgG-degrading enzymes have emerged as innovative therapeutic agents for treating conditions driven by pathogenic antibodies. Here, we used structure-guided rational design to engineer IdeSM33, a double mutant (K167R/D226E) of the IgG-specific bacterial protease IdeS from Streptococcus pyogenes, with improved catalytic efficiency. Biolayer interferometry revealed a fourfold increase in binding affinity relative to wild-type IdeS (IdeSWT). This enhancement is likely attributable to mutations that strengthen hydrogen bonding at the enzyme-IgG Fc interface. In vitro, IdeSM33 has higher performance than IdeSWT in cleaving serum IgG. In vivo studies in rabbits demonstrated that IdeSM33 effectively depleted circulating IgG and showed better performance at a dose of 0.005 mg/kg than the IdeSWT. Although doses greater than 0.2 mg/kg demonstrated higher plasma concentrations of IdeS and a larger AUC 0 to last, they did not show a significant enhancement in the pharmacodynamics of IgG degradation. Importantly, a single dose of IdeSM33 (0.2 mg/kg) potently degraded binding and neutralizing antibodies against AAV9 within 1-2 days and restored hepatic AAV9 transduction in pre-immunized animals. Together, these findings highlight IdeSM33 as a potent and safe engineered enzyme with therapeutic potential for autoimmune disorders, transplant rejection, and overcoming pre-existing humoral immunity in gene therapy.

Cancer immunotherapies rely on tumor-specific T cells, which arise endogenously in most patients with cancer, but can be low frequency and poorly functional. Methods to specifically identify, expand, and manipulate tumor-specific T cells at the rare frequencies found in peripheral blood would enable new immunotherapeutic strategies. Here, we demonstrate an approach to virally transduce polyclonal tumor-reactive T cells across any MHC haplotype and in the absence of knowing the cognate antigen. By generating lentiviral vectors that selectively transduce cells expressing 4-1BB (CD137), a marker of T cell activation, we can transduce antigen-specific T cells with user-defined genetic cargoes that can selectively expand and track individual clonotypes via single-cell sequencing. Anti-4-1BB lentiviruses (4-1BB LVs) encoding therapeutic cargoes can also enhance antigen-specific T cells to extend survival in a xenograft model of human melanoma and transduce tumor-infiltrating T cells from patients with ovarian cancer. Overall, the 4-1BB LV platform targets antigen-specific T cells in a manner agnostic to both the antigen and presenting MHC, with potential applications in adoptive cell therapy manufacturing and TCR identification. One Sentence SummaryEngineered lentiviral vectors targeting 4-1BB selectively activate, expand, and transduce antigen-specific T cells with immunomodulatory cargo.

Protein truncating variants caused by UGA stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases. Suppressor transfer RNA (sup-tRNA) have therapeutic potential for premature termination codon (PTC) repair, but have thus far underperformed by traditional AAV delivery platforms and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to enable payload optimization. These data demonstrate that U6 promotor and AAV2/9 capsids have the lowest in vivo activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100bp genomic context provide broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV delivered ArgUGA sup-tRNA in brain demonstrate no effects on endogenous tRNA levels, their acylation or processing, and these features are also maintained in scAAV delivered ArgUGA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/724978v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1a48274org.highwire.dtl.DTLVardef@170b999org.highwire.dtl.DTLVardef@1a8fdfcorg.highwire.dtl.DTLVardef@1bacb04_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lung is a major site of metastases for many primary cancers associated with poor outcomes. A central challenge in cancer immunotherapy is overcoming tumor immune evasion, which limits effective antitumor responses. Here, we investigated whether combinatorial mRNA-encoded cytokine therapy can overcome tumor immune evasion by coordinately engaging innate and adaptive immunity, using murine models of pulmonary metastases. We employed intravenously administered cationic nucleoside-modified mRNA-lipoplexes (RNA-LPX) for targeted delivery of mRNA-encoded cytokines to the lung. The cytokine mix containing interferon-, half-life extended interleukin (IL)-7, and a half-life extended IL-2 variant with reduced CD25-binding modulated the tumor immune microenvironment resulting in a potent and broad anti-tumor response and prolonged survival with good tolerability at the conditions tested. Using cell depletion experiments, we demonstrated that both T and natural killer (NK) cells are crucial mediators of the observed anti-tumor efficacy of the cytokine RNA mix, which induced activation and effector function of NK and T cells, coupled with reduced regulatory T cells (Treg) numbers and Treg activation in the lung. Importantly, antitumor efficacy was maintained in models of impaired antigen presentation, including loss of an immunodominant tumor antigen and MHC class I deficiency, where NK cells served as the primary effectors. The cytokine RNA mix induced immune cell activation in the primary human lung tumor culture, suggesting potential for translational application. Collectively, these findings demonstrate that combinatorial cytokine therapy can drive both antigen-dependent and antigen-independent tumor control for the treatment of lung metastases.

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.

Oculocutaneous albinism type 1 (OCA1) is a pigmentation disorder caused by biallelic tyrosinase (TYR) mutations, an essential enzyme for melanin synthesis. TYR inactivity results in loss of hair, skin, and eye pigment, which is detrimental for ocular function. Hypopigmentation of iris, retinal pigment epithelium (RPE), and choroid results in severe photosensitivity and low visual acuity. There are currently no FDA-approved pigment restoring therapies for OCA1, making therapeutic development an unmet clinical need. To address this gap, we have advanced an adeno-associated viral (AAV)-mediated Tyr replacement approach for OCA1 ocular pigment restoration. We evaluated the optimal viral delivery strategy and vector cell-type specificity for iris, RPE, and choroid pigmentation in an OCA1 mouse model, testing intraocular and systemic viral delivery methods in conjunction with viral constructs of varying RPE-specificity. Early, systemic delivery of an RPE-directed AAV-Tyr construct, AAV9.2yf-VMD2-Tyr, achieved widespread ocular pigment rescue with minimal off-target expression in non-ocular tissues. Animals treated with AAV9.2yf-VMD2-Tyr demonstrated reduced photophobic behavior compared to untreated controls, indicating that ocular pigmentation restores a debilitating functional consequence of OCA1. Our findings establish a foundation for clinical translation of an AAV-TYR therapy aimed at improving light sensitivity, glare, and low vision through pigment restoration in patients with OCA1.

Inherited retinal diseases (IRDs) are a heterogeneous group of genetic disorders that cause progressive vision loss. A subset of IRDs is associated with ubiquitously expressed genes involved in fundamental cellular processes, often resulting in multisystem disease. Among these is Zellweger spectrum disorder (ZSD), caused by pathogenic variants in PEX genes required for peroxisome biogenesis and function. There are no proven targeted disease-modifying treatments for ZSD, and it is unclear whether localized restoration of peroxisome function is sufficient to mitigate retinal degeneration. We previously demonstrated that HsPEX1 retinal gene augmentation therapy in a mouse model of mild ZSD homozygous for the murine equivalent (PEX1-p.[Gly844Asp]) of the most common deleterious allele in patients (PEX1-c.[2528G>A], PEX1-p.[Gly843Asp]), improved retinal electrophysiological response. Here, we present a comprehensive, dose-range evaluation of a re-designed, clinically relevant AAV8-delivered HsPEX1 subretinal gene therapy, employing expanded outcome measures. We observed a marked improvement in functional vision, retinal response, photoreceptor structure, retinal pigment epithelium integrity, subretinal inflammation, and peroxisomal metabolites, durable to the endpoint of 6 months post single subretinal injection. These studies provide preclinical proof-of-concept that localized retinal gene replacement can mitigate vision loss in peroxisome-mediated IRD.

Personalized neoantigen vaccines are an emerging strategy for cancer immunotherapy, but their effectiveness depends on selecting tumor-specific antigens capable of inducing functional T-cell responses. The VACINUS AI-informed neoantigen prioritization framework previously identified and peptide-validated three immunogenic Tier 1 neoantigens in the B16F10 melanoma model. In this study, we extended that framework by translating these validated neoantigens into a multi-epitope messenger RNA vaccine formulated with lipid nanoparticles and evaluating its preclinical immunogenicity and antitumor activity. The three VACINUS-prioritized B16F10 neoantigens were encoded within a single multi-epitope construct, BF-V1_27-Ser, and formulated to generate BF-RNA-P. In B16F10 tumor-bearing mice, BF-RNA-P induced neoantigen-specific CD8+T-cell responses, with the strongest response directed against the B16F10-1-4 epitope. Combination with anti-PD-1 further enhanced vaccine-induced CD44+IFN-{gamma}+ CD8+ T-cell activation, whereas anti-PD-1 alone did not induce detectable peptide-specific responses. BF-RNA-P also suppressed tumor growth in vivo, and combination treatment produced the strongest antitumor effect, reflected by reduced tumor volume and lower endpoint tumor burden. Together, these findings demonstrate that VACINUS-prioritized and peptide-validated neoantigens can be reformatted into a multi-epitope messenger RNA/lipid nanoparticle vaccine while retaining antigen-specific immunogenicity and antitumor activity. This study provides preclinical proof-of-concept for integrating AI-informed, TCR-aware neoantigen prioritization with messenger RNA/lipid nanoparticle delivery as a translational strategy for personalized cancer vaccine development.

Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of various tumor types by redirecting and activating T cells against tumor cells. However, CAR-T cell manufacturing approaches remain challenging and limit their widespread use in clinical settings. In vivo CAR-T therapy bypasses ex vivo cell manufacturing and patient preconditioning limitations; however, it faces a significant safety concern as CAR proteins on viral packaging cells are incorporated into budding virions, leading to off-target transduction of tumor cells. Here, we address this risk by developing the CAR-Less ER-Anchor Vector (CLEAN-V) system. By exploiting endoplasmic reticulum (ER) retention, CLEAN-V prevents the CAR protein from trafficking to the cell surface during viral packaging, thereby blocking its incorporation into the viral envelope. CLEAN-V particles exhibit near-complete loss of CAR-mediated tumor cell transduction. Furthermore, CLEAN-V integrates seamlessly into existing third-generation LVV workflows in four- or five-plasmid formats and generates CAR-T cells with preserved phenotypic and functional integrity. These results establish CLEAN-V as a robust platform for developing safe, targeted lentiviral vectors for in vivo CAR-T therapy.

Pathogenic variants in ATAD3A cause a spectrum of multisystem disorders, with a recurrent dominant-negative variant (c.1582C>T; p.Arg528Trp) associated with neurodevelopmental disease. Given the tolerance of ATAD3A to heterozygous loss of function variants, allele-specific transcript reduction represents a promising therapeutic strategy. We designed and optimized allele-specific antisense oligonucleotides (ASOs) targeting the c.1582C>T transcript and evaluated their efficacy and specificity in affected fibroblasts using allele-specific primers and amplicon-based next generation sequencing. Therapeutic potential was further assessed in vivo in zebrafish embryos expressing human wild-type or mutant ATAD3A transcripts. An optimized gapmer ASO selectively reduced mutant ATAD3A transcripts while relatively sparing the wild-type allele. In addition to RNase H-mediated degradation, the ASO induced exon skipping, leading to degradation of the aberrant transcript without production of a truncated protein. In zebrafish, expression of mutant human ATAD3A in embryos caused developmental abnormalities including reduced eye size, which were robustly rescued by co-injection of the optimized ASO. Our findings provide proof of concept for allele-targeted ASO therapy for dominant-negative ATAD3A variants. This work highlights the therapeutic potential of ASOs for rare dominant disorders involving genes tolerant to heterozygous loss-of-function, and establishes zebrafish as a versatile platform for in vivo ASO optimization.

Efficient delivery of genetic cargo to primary human T cells remains a critical barrier for cell-based immunotherapies. AAVs are widely used but limited by immune neutralization and poor T cell transduction efficiency. We present NEO-AAV, an engineered fusion protein (PH-ALG2-PKD12) that recruits AAV capsids into endogenous extracellular vesicles (EVs) via PI(4,5)P2-directed membrane targeting, multivalent PKD12-capsid clustering, and ALG2-mediated ESCRT machinery recruitment, yielding EV-enveloped particles with improved AAV loading compared with passive EV-AAV controls. NEO-AAV displayed enhanced resistance to an anti-AAV6 neutralizing antibody (ADK6), maintaining transduction at concentrations that neutralized naked AAV6 and outperformed passively formed EV-AAV6. Surface display of a CD7/CD3/CD28 tri-chimera enabled single-step activation and transduction of CD7 T cells within PBMCs, reaching [~]38% eGFP cells without exogenous pre-activation. As a proof-of-concept, NEO-AAV generated functional CAR-T cells from primary human PBMCs, exhibiting antigen-specific IFN-{gamma} release and cytotoxicity against CD19+ target cells. CAR expression peaked at day 5 and declined by day 15, consistent with episomal AAV kinetics, framing NEO-AAV as an activation-coupled delivery module rather than a durable solution. Together, NEO-AAV provides a programmable EV-enveloped AAV platform with improved immune shielding and single-step T cell transduction, offering a building block for immune cell engineering with in vivo potential.

Pioneering research is adapting chimeric antigen receptors (CARs) from oncology to Alzheimers disease (AD) by targeting amyloid beta (A{beta}). Newer synthetic receptor systems can go beyond, transforming cells into targeted biological drug factories that can couple A{beta} detection to synthesis and secretion of genetically encoded therapeutics. Among candidate systems, T cells Redirected for Universal Cytokine Killing (TRUCK), synthetic Notch (synNotch), and Synthetic Intramembrane Proteolysis Receptors (SNIPR) have shown promise in oncology. Here, we adapt these platforms to AD using a shared A{beta}-targeting binding domain derived from Aducanumab (Aduhelm), coupled to inducible expression cassettes driving identical transgenes: secreted Metridia luciferase (MetLuc) and a Lecanemab (Leqembi)-based chimeric human-mouse antibody (chLecanemab). To validate these systems in vitro, Jurkat clones expressing each receptor were treated with oligomer-enriched A{beta} (A{beta}O) to model AD, and receptor output was quantified by media MetLuc levels and chLecanemab colocalization with A{beta} aggregates. For TRUCK systems, we show the A{beta}-targeting CAR successfully activated Jurkat cells by flow cytometry. We also show that six Nuclear Factor of Activated T-cells (NFAT) tandem repeat response elements (6xNFAT) paired with either minimal interleukin-2, synthetic TATA box, or minimal cytomegalovirus promoters resulted in functional regulatory regions. Despite this, all TRUCK variants failed to significantly upregulate MetLuc in response to A{beta}O. In contrast, both synNotch and SNIPR responded robustly to A{beta}O, with SNIPR outperforming synNotch in both MetLuc and chLecanemab production. These findings establish SNIPR and synNotch as promising platforms for future research on cell-based targeted therapeutic delivery in AD.

Adeno-associated virus (AAV)-delivered anti-HIV-1 broadly neutralizing antibodies (bNAbs) have demonstrated promise for preventing and treating HIV-1 infection in preclinical models. However, host immune responses, specifically anti-drug antibodies (ADA), limit sustained bNAb expression. We have previously shown that PD-L1-mediated immune shielding improves the consistency of AAV-delivered bNAb 3BNC117 expression from muscle tissue in rhesus macaques. Here, we test the breadth of this approach with another bNAb, 10-1074. We show that AAV9.PD-L1 co-delivery with AAV9.10-1074 reduced the occurrence of ADA responses and improved the durability of bNAb expression for one year post administration. Notably 12 of 12 macaques that received AAV9.10-1074 vectors were protected against ten repeated SHIVAD8-EO challenges. Histopathological profiling showed that AAV9.PD-L1 co-delivery prevented severe local inflammation and tertiary lymphoid structure formation at the administration site. Thus, immune shielding could serve as a broad strategy to prolong transgene expression from muscle-directed AAV-delivered biologics.

BackgroundBiallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinsons disease (EOPD). Parkin is a crucial component of PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration. ObjectiveInvestigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. MethodsWe initially validated phosphorylated ubiquitin Ser65 (pUbSer65) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated PRKN replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. ResultsOur research showed pUbSer65 signal was reduced in Parkin-KO SH-SY5Y cells when compared to wild-type cells after mitochondrial stress, indicating deficiency in initiation of mitophagy. AAV-mediated human PRKN gene replacement successfully restored these pUbSer65 levels in knockout cells. We saw restoration in patient-derived fibroblasts following AAV-Parkin overexpression. We developed a translatable gene therapy approach using rodents. We demonstrated the feasibility of delivering AAV-Parkin directly into the substantia nigra (SN) of wild-type rats. Using an AAV1 capsid with Ef1a promoter, we achieved dose-dependent Parkin expression and identified a well-tolerated dose. We also evaluated multiple promoters in a proprietary Spark100 capsid, finding Ef1a and Synapsin1 (Syn1) were most effective for transducing dopaminergic neurons in the SN of mice without causing adverse effects. These findings established a well-tolerated vector dose and an optimal capsid-promoter combination. ConclusionsOur results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/737487v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16dd13corg.highwire.dtl.DTLVardef@c3dfcdorg.highwire.dtl.DTLVardef@19a310dorg.highwire.dtl.DTLVardef@a66f2_HPS_FORMAT_FIGEXP M_FIG C_FIG