Adeno-associated virus as a delivery vector for gene therapy of human diseases.

Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.

Integration and the risk of liver cancer-Is there a real risk?

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Retention Rates of Genetic Therapies Based on AAV Serotypes 2 and 8 Using Different Drug-Delivery Materials.

The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip® cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip® cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip® cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.

Gene replacement therapy in Bietti crystalline corneoretinal dystrophy: an open-label, single-arm, exploratory trial.

Bietti crystalline corneoretinal dystrophy is an inherited retinal disease caused by mutations in CYP4V2, which results in blindness in the working-age population, and there is currently no available treatment. Here, we report the results of the first-in-human clinical trial (NCT04722107) of gene therapy for Bietti crystalline corneoretinal dystrophy, including 12 participants who were followed up for 180-365 days. This open-label, single-arm exploratory trial aimed to assess the safety and efficacy of a recombinant adeno-associated-virus-serotype-2/8 vector encoding the human CYP4V2 protein (rAAV2/8-hCYP4V2). Participants received a single unilateral subretinal injection of 7.5 × 1010 vector genomes of rAAV2/8-hCYP4V2. Overall, 73 treatment-emergent adverse events were reported, with the majority (98.6%) being of mild or moderate intensity and considered to be procedure- or corticosteroid-related; no treatment-related serious adverse events or local/systemic immune toxicities were observed. Compared with that measured at baseline, 77.8% of the treated eyes showed improvement in best-corrected visual acuity (BCVA) on day 180, with a mean ± standard deviation increase of 9.0 ± 10.8 letters in the 9 eyes analyzed (p = 0.021). By day 365, 80% of the treated eyes showed an increase in BCVA, with a mean increase of 11.0 ± 10.6 letters in the 5 eyes assessed (p = 0.125). Importantly, the patients' improvement observed using multifocal electroretinogram, microperimetry, and Visual Function Questionnaire-25 further supported the beneficial effects of the treatment. We conclude that the favorable safety profile and visual improvements identified in this trial encourage the continued development of rAAV2/8-hCYP4V2 (named ZVS101e).

[Adeno-associated virus-mediated hepatocyte-specific NDUFA13 overexpression protects against CCl4-induced liver fibrosis in mice by inhibiting hepatic NLRP3 activation].

OBJECTIVE: To investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms. METHODS: BALB/C mice (7 to 8 weeks old) were divided into normal group, CCl4 group, CCl4+AAV-NC group and CCl4+AAV-NDU13 group (n=18). Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4 twice a week for 3, 5 or 7 weeks, and the recombinant virus AAV8-TBG-NC or AAV8-TBG-NDUFA13 was injected via the tail vein 7-10 days prior to CCl4 injection. After the treatments, pathological changes in the liver of the mice were observed using HE and Masson staining. Hepatic expression levels of NDUFA13 and α-SMA were detected with Western blotting, and the coexpression of NDUFA13 and NLRP3, TNF-α and IL-1ß, and α-SMA and collagen Ⅲ was analyzed with immunofluorescence assay. RESULTS: HE and Masson staining showed deranged liver architecture, necrotic hepatocytes and obvious inflammatory infiltration and collagen fiber deposition in mice with CCl4 injection (P < 0.001). NDUFA13 expression markedly decreased in CCl4-treated mice (P < 0.001), while a significant reduction in inflammatory aggregation and fibrosis was observed in mice with AAV-mediated NDUFA13 overexpression (P < 0.001). In CCl4+AAV-NDU13 group, immunofluorescence assay revealed markedly weakened activation of NLRP3 inflammasomes (P < 0.001), significantly decreased TNF-α and IL-1ß secretion (P < 0.001), and inhibited hepatic stellate cell activation (P < 0.05) and collagen formation in the liver (P < 0.001). CONCLUSION: Mitochondrial NDUFA13 overexpression in hepatocytes protects against CCl4- induced liver fibrosis in mice by inhibiting activation of NLRP3 signaling.

Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production.

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.

Using an In Vivo Mouse Model to Determine the Exclusion Criteria of Preexisting Anti-AAV9 Neutralizing Antibody Titer of Pompe Disease Patients in Clinical Trials.

The efficacy of adeno-associated virus (AAV)-based gene therapy is dependent on effective viral transduction, which might be inhibited by preexisting immunity to AAV acquired from infection or maternal delivery. Anti-AAV neutralizing Abs (NAbs) titer is usually measured by in vitro assay and used for patient enroll; however, this assay could not evaluate NAbs' impacts on AAV pharmacology and potential harm in vivo. Here, we infused a mouse anti-AAV9 monoclonal antibody into Balb/C mice 2 h before receiving 1.2 × 1014 or 3 × 1013 vg/kg of rAAV9-coGAA by tail vein, a drug for our ongoing clinical trials for Pompe disease. The pharmacokinetics, pharmacodynamics, and cellular responses combined with in vitro NAb assay validated the different impacts of preexisting NAbs at different levels in vivo. Sustained GAA expression in the heart, liver, diaphragm, and quadriceps were observed. The presence of high-level NAb, a titer about 1:1000, accelerated vector clearance in blood and completely blocked transduction. The AAV-specific T cell responses tended to increase when the titer of NAb exceeded 1:200. A low-level NAbs, near 1:100, had no effect on transduction in the heart and liver as well as cellular responses, but decreased transduction in muscles slightly. Therefore, we propose to preclude patients with NAb titers > 1:100 from rAAV9-coGAA clinical trials.

Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights.

Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in molecular engineering, high-throughput screening platforms, and computational techniques have resulted in a toolbox of capsid variants with enhanced performance over parental serotypes. Despite their considerable promise and emerging clinical success, there are still obstacles hindering their broader use, including limited transduction capabilities, tissue/cell type-specific tropism and penetration into tissues through anatomical barriers, off-target tissue biodistribution, intracellular degradation, immune recognition, and a lack of translatability from preclinical models to clinical settings. Here, we first describe the transduction mechanisms of natural AAV serotypes and explore the current understanding of the systemic and cellular hurdles to efficient transduction. We then outline progress in developing designer AAV capsid variants, highlighting the seminal discoveries of variants which can transduce the central nervous system upon systemic administration, and, to a lesser extent, discuss the targeting of the peripheral nervous system, eye, ear, lung, liver, heart, and skeletal muscle, emphasizing their tissue and cell specificity and translational promise. In particular, we dive deeper into the molecular mechanisms behind their enhanced properties, with a focus on their engagement with host cell receptors previously inaccessible to natural AAV serotypes. Finally, we summarize the main findings of our review and discuss future directions.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio.

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases in vivo.

Locally Directed Recombinant Adeno- Associated Virus-Mediated IGF-1 Gene Therapy Enhances Osteochondral Repair and Counteracts Early Osteoarthritis In Vivo.

BACKGROUND: Restoration of osteochondral defects is critical, because osteoarthritis (OA) can arise. HYPOTHESIS: Overexpression of insulin-like growth factor 1 (IGF-1) via recombinant adeno-associated viral (rAAV) vectors (rAAV-IGF-1) would improve osteochondral repair and reduce parameters of early perifocal OA in sheep after 6 months in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral defects were created in the femoral trochlea of adult sheep and treated with rAAV-IGF-1 or rAAV-lacZ (control) (24 defects in 6 knees per group). After 6 months in vivo, osteochondral repair and perifocal OA were assessed by well-established macroscopic, histological, and immunohistochemical scoring systems as well as biochemical and micro-computed tomography evaluations. RESULTS: Application of rAAV-IGF-1 led to prolonged (6 months) IGF-1 overexpression without adverse effects, maintaining a significantly superior overall cartilage repair, together with significantly improved defect filling, extracellular matrix staining, cellular morphology, and surface architecture compared with rAAV-lacZ. Expression of type II collagen significantly increased and that of type I collagen significantly decreased. Subchondral bone repair and tidemark formation were significantly improved, and subchondral bone plate thickness and subarticular spongiosa mineral density returned to normal. The OA parameters of perifocal structure, cell cloning, and matrix staining were significantly better preserved upon rAAV-IGF-1 compared with rAAV-lacZ. Novel mechanistic associations between parameters of osteochondral repair and OA were identified. CONCLUSION: Local rAAV-mediated IGF-1 overexpression enhanced osteochondral repair and ameliorated parameters of perifocal early OA. CLINICAL RELEVANCE: IGF-1 gene therapy may be beneficial in repair of focal osteochondral defects and prevention of perifocal OA.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges.

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.

Current and emerging gene therapies for haemophilia A and B.

INTRODUCTION: After decades of stumbling clinical development, the first gene therapies for haemophilia A and B have been commercialized and have normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several other clinical programs testing adeno-associated viral (AAV) vector gene therapy are at various stages of clinical testing. DISCUSSION: Multiyear follow-up in phase 1/2 and 3 studies showed long-term and sometimes curative but widely variable and unpredictable efficacy. Liver toxicities, mostly low-grade, occur in the 1st year in at least some individuals in all haemophilia A and B trials and are poorly understood. Wide variability and unpredictability of outcome and slow decline of FVIII levels are a major disadvantage because immune responses to AAV vectors preclude repeat dosing, which otherwise could improve suboptimal or restore declining expression, while overexpression may predispose to thrombosis. Long-term safety outcomes will need lifelong monitoring because AAV vectors infused at high doses integrate into chromosomes at rates that raise questions about potential oncogenicity and necessitate vigilance. Alternative gene transfer systems employing gene editing and/or non-viral vectors are under development and promise to overcome some limitations of the current state of the art for both haemophilia A and B. CONCLUSIONS: AAV gene therapies for haemophilia have now become new treatment options but not universal cures. AAV is a powerful but imperfect gene transfer platform. Biobetter FVIII transgenes may help solve some problems plaguing gene therapy for haemophilia A. Addressing variability and unpredictability of efficacy, and delivery of gene therapy to ineligible patient subgroups may require different gene transfer systems, most of which are not ready for clinical translation yet but bring innovations needed to overcome the current limitations of gene therapy.

Studying the ssDNA loaded adeno-associated virus aggregation using coarse-grained molecular dynamics simulations.

The aggregation of adeno-associated viral (AAV) capsids in an aqueous environment was investigated via coarse-grained molecular dynamics (CG-MD) simulations. The primary driving force and mechanism of the aggregation were investigated with or without single-strand DNA (ssDNA) loaded at various process temperatures. Capsid aggregation appeared to involve multiple residue interactions (i.e., hydrophobic, polar and charged residues) leading to complex protein aggregation. In addition, two aggregation mechanisms (i.e., the fivefold face-to-face contact and the edge-to-edge contact) were identified from this study. The ssDNA with its asymmetric structure could be the reason for destabilizing protein subunits and enhancing the interaction between the charged residues, and further result in the non-reversible face-to-face contact. At higher temperature, the capsid structure was found to be unstable with the significant size expansion of the loaded ssDNA which could be attributed to reduced number of intramolecular hydrogen bonds, the increased conformational deviations of protein subunits and the higher residue fluctuations. The CG-MD model was further validated with previous experimental and simulation data, including the full capsid size measurement and the capsid internal pressure. Thus, a good understanding of AAV capsid aggregation, instability and the role of ssDNA were revealed by applying the developed computational model.

Capsid-mediated control of adeno-associated viral transcription determines host range.

Adeno-associated virus (AAV) is a member of the genus Dependoparvovirus, which infects a wide range of vertebrate species. Here, we observe that, unlike most primate AAV isolates, avian AAV is transcriptionally silenced in human cells. By swapping the VP1 N terminus from primate AAVs (e.g., AAV8) onto non-mammalian isolates (e.g., avian AAV), we identify a minimal component of the AAV capsid that controls viral transcription and unlocks robust transduction in both human cells and mouse tissue. This effect is accompanied by increased AAV genome chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that host factors are selectively recruited by the VP1 N terminus of AAV8 but not avian AAV. Notably, these include AAV essential factors implicated in the nuclear factor κB pathway, chromatin condensation, and histone methylation. We postulate that the AAV capsid has evolved mechanisms to recruit host factors to its genome, allowing transcriptional activation in a species-specific manner.

Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.