Molecular Therapy - Methods & Clinical Development

In gene therapy, delivery vectors are a key component for successful gene delivery and safety, based on which adeno-associated viruses (AAVs) gained popularity in particular for the liver, but also for other organs. Traditionally, rodents have been used as animal models to develop and optimize treatments, but species and organ specific tropism of AAV desire large animal models more closely related to humans for preclinical in-depth studies. Relevant AAV variants with the potential for clinical translation in liver gene therapy were previously evolved in vivo in a xenogeneic mouse model transplanted with human hepatocytes. Here, we selected and evaluated efficient AAV capsids using chimeric mice with a >90% xenografted pig hepatocytes. The pig is a valuable preclinical model for therapy studies due to its anatomic and immunological similarities to humans. Using a DNA-barcoded recombinant AAV library containing 47 different capsids and subsequent Illumina sequencing of barcodes in the AAV vector genome DNA and transcripts in the porcine hepatocytes, we found the AAVLK03 and AAVrh20 capsid to be the most efficient delivery vectors regarding transgene expression in porcine hepatocytes. In attempting to validate these findings with primary porcine hepatocytes, we observed capsid-specific differences in cell entry and transgene expression efficiency where the AAV2, AAVAnc80, and AAVDJ capsids showed superior efficiency to AAVLK03 and AAVrh20. This work highlights intricacies of in vitro testing with primary hepatocytes and the requirements for suitable pre-clinical animal models but suggests the chimeric mouse to be a valuable model to predict AAV capsids to transduce porcine hepatocytes efficiently.

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

Determining the concentration of recombinant adeno-associated virus (AAV) productions, also known as titering, is crucial not only for quality control purposes but also for comparative studies of preclinical and clinical gene therapy trials. Recently, several AAVs were engineered by inserting seven amino acids at the outermost tip of the capsids protruding VR-VIII loop. These variants have demonstrated increased transduction capabilities over naturally occurring AAV serotypes in several studies. However, they have also been shown to produce lower yields when titered using standard techniques, raising questions about their adequacy for clinical development and use. Here, we investigated why peptide insertion onto AAV capsids reduces their titer by examining viral stocks using electron microscopy and PCR-based titering. We reveal that the DNAse digestion step, performed to eliminate free-floating DNA prior to qPCR or ddPCR, adversely impacts engineered capsid stability due to exposure to heat, artificially lowering viral titers of engineered serotypes. Titering without heating yields significantly higher titers for these variants which have melting temperatures (Tm) close to the DNAse inactivation temperature, while titers for parental serotypes with higher Tm remain unchanged. Our findings provide an important new perspective for titering engineered variants with lower thermostability, especially when comparing their effectiveness to their parental serotypes.

Recombinant Adeno-associated viral vector (rAAV) mobilization is a largely theoretical process in which intact AAV vectors spread or "mobilize" from transduced cells and infect additional cells within, or external, of the initial host. This process can be replication independent (vector alone), or replication-dependent (de novo rAAV production facilitated by super-infection of both wild-type AAV (wtAAV) and Ad helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 Kb), rAAV-IDUA (3.7 Kb), and rAAV-NanoDysferlin (4.9 Kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and a rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be de fined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed non-replicative mobilization). In rAAV infected cells co-infected with Ad5 and wtAAV, rAAV particle production was increased > 50-fold compared to non-replicative conditions. In addition, replicative dependent rAAV vectors mobilized and resulted in >1,000 -fold transduction upon a subsequent 2nd round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization resistant vectors and the opportunity to derive better vector production systems.

Adeno-associated virus purification by density-gradient ultracentrifugation is labor-intensive and often results in substantial titer loss due to particle aggregation. Here, we present a scalable co-isolation strategy in which AAV is precipitated together with extracellular vesicles secreted by the producer cell line, completely bypassing density-gradient separation. The resulting AAV-EV preparations comprise free AAV, free EVs, and EV-associated AAV. Functionally, AAV-EV vectors (AAV2/1 serotype) support efficient ex vivo genome editing across multiple independent loci in mouse and rat zygotes, achieving a mean targeting efficiency of approximately 26%. Compared with gradient-purified AAV administered at matched doses, AAV-EV formulations yielded 2.34-fold higher embryo viability while maintaining equivalent transgene copy numbers. By leveraging EVs as a biological matrix, this approach enables ultracentrifugation-free AAV isolation without compromising vector functionality. Overall, AAV-EV represents an accessible and embryo-tolerant platform for rodent genome engineering that aligns with the principles of Replacement, Reduction, and Refinement (3R) principles.

Though cell therapy for cancer is now widely used commercially and has been efficacious for tens of thousands of patients, conventional manufacturing methods are expensive and difficult to scale. An alternative approach is to deliver the relevant anti-tumor transgenes to T cells in vivo in the patient. Such "in vivo cell therapy" methods promise to be more scalable, with reduced cost of goods, since the same drug product can be administered to any patient. Typically, conventional cell therapy introduces anti-tumor transgenes into T cells using a lentivector pseudotyped with VSV-G. However, VSV-G is not cell type specific because its molecular target is present on a diversity of human cells, which may result in less than optimal pharmacology in vivo. Natures existing viral diversity presents the opportunity to identify alternative pseudotypes that are more optimal for in vivo cell therapy. In this study, we first performed a large-scale bioinformatic sequence search for G-proteins similar to VSV-G. We identified 166 G-proteins in the sequence search and then tested 9 in vitro for efficiency, specificity, and sensitivity of transgene delivery to various human immune cell phenotypes. We used the results from this screen to select three G-protein candidates for a pilot GFP transgene delivery study in humanized mice, using anti-CD3 antibody fragments for T cell tropism. One candidate G-protein performed significantly better than VSV-G, so we moved that candidate into tumor control studies in a humanized mouse model. This candidate G-protein was able to deliver an anti-tumor transgene to T cells, which subsequently cleared 100% of tumor burden in 100% of mice. We conclude that systematic screens for optimal lentivector designs can be used to identify optimized candidates for in vivo cell therapy.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

Limited infiltration capacity significantly limits the effectiveness of chimeric antigen receptor (CAR) T cells for solid tumors. We have recently developed a large family of highly modular synthetic cytokine receptors termed velocity receptors (VRs), capable of binding key inflammatory cytokines, such as IL5, IL8, and TNF, which drive CAR T cells into an elevated motility state. These new CAR T cells sense and amplify these autocrine secreted cytokines, thereby maintaining a self-propelled, high migratory state, facilitating penetration into dense tumor cores. In this study, we systematically evaluated key factors influencing VR transduction in order to improve their stable integration and expression. We established a dual-fluorescence reporter system to allow simultaneous monitoring of both VR and CAR constructs, and while evaluating modifications to the vector construct and generating standardized infectious unit (IFU) curves under various conditions. Our results demonstrate that the attempt to reduce overall lentiviral vector size by eliminating non coding sections upstream of the central polypurine tract (cPPT) do not yield better transduction efficiency, though it is unclear if the effect is due to viral production or integration impairment. We also observed a log-linear relationship between viral dose and transduction efficiency for a subset of VRs previously tested in various mouse models of human cancer, with VR5IL8 and VR5TNF VRs consistently outperforming VR5IL5 and V5 (full length native IL5 receptor). Overall, these findings establish an optimized and reproducible framework that offers valuable guidance for the future development and functional study of VR-CAR T cells in cellular therapies for solid tumors.

Efficient systemic delivery to the lung remains a major barrier for adeno-associated virus (AAV)-mediated pulmonary gene therapy, particularly when pre-existing immunity limits the use of conventional capsids. Here, we evaluated Bovine AAV, a phylogenetically divergent capsid, as candidate vector for lung-directed gene transfer. In adult C57BL/6J mice, intravenous delivery of Bovine AAV resulted in robust and preferential lung transduction comparable to AAV4, with predominant targeting of alveolar type I pneumocytes and pulmonary endothelial cells. In primary human lung-resident cells, Bovine AAV was particularly effective in microvascular endothelial cells, a target poorly transduced by AAV4 in vitro. Bovine AAV demonstrated scalable production with yield, purification performance, capsid quality, and genome integrity comparable to AAV9. In sera from healthy adults from the United States and Switzerland, Bovine AAV showed intermediate neutralization frequencies, lower than AAV2 and AAV4 but higher than AAV5 and AAV9. Of relevance, Bovine AAV maintained in vivo transduction efficiency in mice previously immunized with a pool of human and non-human primate-derived AAV capsids, including AAV4. Together, these results position Bovine AAV as a promising lung-tropic and immune-distinct vector for pulmonary gene therapy, with particular relevance for applications requiring systemic delivery in the presence of pre-existing immunity to conventional serotypes.

Adeno-associated virus (AAV) vectors are widely used for gene delivery, but inefficient transduction can require high vector doses. We tested whether unmodified linear polyvinylpyrrolidone (PVP), a pharmaceutical excipient, can improve AAV formulation without chemical modification of the vector or polymer. PVP10, PVP40, and PVP360 were evaluated in vitro across HEK293, HeLa, MEF, and CHO cells; selected formulations were tested after intravenous delivery, and 3% PVP40 was tested by subretinal delivery. In vitro, 1-3% PVP increased AAV8- and AAV9-mediated GFP expression across multiple cell lines. PVP360 showed broad activity in the initial cross-cell assay, whereas the HEK293 molecular-weight screen identified PVP40 and PVP360 as the most active formulations. The substantial fold increase observed in CHO cells largely reflected the low baseline transduction of the control group. MTT absorbance declined with concentrated PVP360, whereas PVP40 retained transduction-enhancing activity and was selected for local testing. In the HeLa AAV-DJ assay, the response pattern differed between MOI 1,000 and MOI 100; the largest observed increases in GFP-positive area occurred with 1.5% PVP10 and 1.5% PVP360 at MOI 100. Intravenous AAV9 delivery with PVP did not consistently increase ex vivo organ reporter signal. By contrast, subretinal delivery of AAV-PHP.eB with 3% PVP40 produced a 1.79- fold larger mean DsRed-positive area per retinal section, averaged within each eye (Welchs t-test p = 0.063; Bayesian Pr[{Delta} > 0] = 0.952). These findings support further evaluation of PVP40 for local subretinal AAV delivery.

Although adeno-associated virus (AAV) has enjoyed enormous success as a delivery modality for gene therapy, it continues to suffer from the high prevalence of preexisting neutralizing antibodies in human populations, limiting who can receive potentially life-saving treatments. In this regard, AAV therapies generally also must be administered as a single dose since neutralizing antibodies develop in patients who receive the virus. Strategies for circumventing these issues remain limited. As a novel solution, we employed SpyTag-SpyCatcher molecular glue technology to facilitate packaging of AAVs inside of recombinant protein vault nanoparticles. Vaults are endogenous particles produced by mammalian cells. We therefore hypothesized that they may shield packaged molecules from neutralizing antibodies. Vaults have previously been utilized to deliver drugs and proteins into cells, but our study represents the first time anyone has packaged an entire virus inside of a vault. We showed that our vaultAAV (VAAV) delivery vehicle transduces cells in the presence of anti-AAV neutralizing serum. VAAV is positioned as a new gene therapy delivery platform with potential to overcome the neutralizing antibody problem and perhaps even allow administration of multiple doses, expanding the scope of AAV treatments.

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.

Adeno-associated viruses (AAVs) have been used in gene therapy, especially for inherited retinal diseases. Despite their effectiveness in gene transduction, immune responses to the AAV capsid and transgene products have been reported, which can compromise both the efficacy and safety of AAV-mediated therapies. The eye is regarded as an immune-privileged organ where immune activity is constitutively suppressed. Here, we highlight that immunomonitoring in an ocular gene transfer reveals variable immune responses, whatever the species (human clinical trial, non-human primates, mice), the site of injection, the cassette, and the dose. We further explored factors contributing to this variability, investigating the correlation among immune parameters in a controlled experimental setting. In a syngeneic murine model after an intraocular injection of AAV, our results highlight an inter-individual variability of immune parameters, emphasizing the importance of considering inherent variability among individuals while designing personalized therapies.

Recombinant adeno-associated virus (rAAV) vectors show therapeutic potential, but their biomanufacturing is limited by low yields and high costs. Host cell-cycle modulation is emerging as a promising strategy to enhance rAAV production. Two G2/M phase-arresting small molecules, ABT-751, a microtubule inhibitor, and helenalin, a thiol-reactive sesquiterpene lactone, were applied post-transfection in HEK293 cells to evaluate how cell-cycle arrest and stress pathways influence rAAV yields. ABT-751 induced G2/M arrest with minimal cytotoxicity, leading to a near five-fold increase in rAAV vector genomes across multiple serotypes and production platforms. Helenalin caused G2/M arrest, yet suppressed rAAV production. Comparative transcriptomic profiling (RNA-Seq) revealed that helenalin altered expression of a widespread set of genes (4,579) compared to control, characterized by rampant p53, ferroptosis, and endoplasmic reticulum dysregulation that overflowed into unfolded protein response with CHOP induction and apoptosis. ABT-751 elicited a more moderate, targeted response (1,895 differentially expressed genes) in a similar subset of pathways, including compensatory mechanisms mitigating oxidative stress. Together, these findings indicate that cell-cycle arrest alone is insufficient to improve rAAV yield. Indeed, tailored cell-cycle modulation, coupled with balanced activation of cellular stress pathways, can enhance rAAV manufacturing efficiency, facilitating more scalable and cost-effective gene therapy production strategies for the future.

Hematopoietic stem cells (HSC) are important targets for gene modification therapies (GMT) as they originate several serious genetic conditions including the {beta}-haemoglobinopathies. Potentially curative ex vivo GMT pose the barriers of accessibility, myeloablation-associated morbidity and prohibitive cost. In vivo GMT using non-integrating single-strand adeno-associated viral vectors (ssAAV) are a promising alternative that address these challenges directly, although the small ssAAV payload limits the capacity to package much larger gene or base editors. We investigated the feasibility of targeting human HSC in vivo with a dual-ssAAV6 strategy, which in future may be useful to deliver split-intein editing tools to overcome this limitation. We engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ humice with human hCD45+CD34+ HSC from transfusion-dependent {beta}-thalassemic patients to test in vivo targeting of hCD45 cells with ssAAV6, then administered 5E+12 genomes/kg of ssAAV6-GFP/ssAAV6-mCherry. Humice showed peak single-transgene expression (GFP+ or mCh+) of 1.96-10.17%, and dual-transgene expression (GFP+mCh+) of 31.77% in circulating hCD45+ cells. Nested hCD45+ from liver, spleen and bone marrow showed single-and dual-transgene expression of 36.13-68.14% and 21.91-59.44% respectively. Secondary transplantation experiments demonstrated long-term persistence of AAV6-transduced hCD45 cells showing single-and dual-transgene expression of 9.19-60.72% and 7.15-9.19% respectively, with significant increase in expression from circulating cells. Minimal pro-inflammatory cytokine expression was observed following ssAAV6 administration in thalassemia humice compared with humice carrying non-thalassemia HSC. Our model demonstrates the efficiency of in vivo ssAAV6-mediated targeting of thalassaemia HSC, potential long-term survivability of transduced cells, and feasibility of a dual-AAV strategy for gene editing, which offers a promising alternative to ex vivo GMT for {beta}-haemoglobinopathies.

Human-derived cortical organoids (hCOs) recapitulate cell diversity and 3D structure found in the human brain and offer a promising model for discovery of new gene therapies targeting neurological disorders. Adeno-associated viruses (AAVs) are the most promising vehicles for non-invasive gene delivery to the central nervous system (CNS), but reliable and reproducible in vitro models to assess their clinical potential are lacking. hCOs can take on these issues as they are a physiologically relevant model to assess AAV transduction efficiency, cellular tropism, and biodistribution within the tissue parenchyma, all of which could significantly modulate therapeutic efficacy. Here, we examine a variety of naturally occurring AAV serotypes and measure their ability to transduce neurons and glia in hCOs from multiple donors. We demonstrate cell tropism driven by AAV serotype and hCO donor and quantify fractions of neurons and astrocytes transduced with GFP as well as overall hCO health.

The blood-brain barrier (BBB) severely restricts the delivery of systemically administered adeno-associated virus (AAV) vectors for central nervous system (CNS) gene therapy. To overcome this limitation, we engineered a library of AAV9 capsid variants through rational design focused on the low-density lipoprotein receptor-related protein 6 (LRP6), a conserved mediator of transcytosis. A multi-tiered screening strategy, encompassing human BBB endothelial cells followed by neuronal and glial target cells in vitro, identified three lead variants (QL9-21, QL9-22, and QL9-25) with markedly enhanced transduction potential. In mice, these variants achieved a 5-28 fold increase in brain-wide gene delivery compared to AAV9, without elevating hepatic tropism. Crucially, evaluation in non-human primates (NHPs) revealed that the lead variant, QL9-21, mediated a striking 3-40 fold enhancement in viral genome delivery across all examined brain regions versus AAV9, while concurrently reducing liver accumulation by 2.6 fold. Our study establishes an LRP6-guided engineering platform that yields novel AAV9 vectors capable of efficient, species-conserved BBB penetration coupled with a favorable safety profile, representing a significant advance toward clinically translatable CNS gene therapies.

Adeno-associated virus (AAV) gene therapies have achieved some clinical success, with multiple products reaching regulatory approval. Encapsulation of AAV vectors within engineered extracellular vesicles (EVs) is an emerging strategy which could help overcome challenges including pre-existing anti-capsid immunity and the need for controlling targeting and tropism. To guide the development of EV-AAV technologies, we developed an assay for quantifying and controlling for the contribution of pseudotransduction to evaluations of EV-AAV-mediated transduction. We developed an AAV vector that switches its transgene output from one reporter to another when acted upon by Cre recombinase expressed in a recipient cell. Using this platform, we investigated EV-AAV transduction as a function of various engineered EV surface modifications. For actively endocytic cells (HEK293FTs), modifications that enhance EV uptake and membrane fusion influence pseudotransduction but not true transduction. Conversely, in less endocytic Jurkat T cells, modifications enhancing EV uptake enhanced both pseudotransduction and true transduction. These conclusions held across two AAV serotypes. Our results provide new insight into prior reports and suggest that effects of enhancing uptake and membrane fusion of EV-AAV vectors are recipient cell type-specific. The methods developed here unambiguously dissect EV-AAV transduction mechanisms and can guide future bioengineering of EV-AAV vectors.

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

Recombinant adeno-associated virus (rAAV) vectors are widely used for gene delivery but show limited efficiency in immune cells, including T lymphocytes and natural killer (NK) cells. To overcome this barrier, we developed a modular rAAV vector engineering strategy that integrates capsid retargeting with genome optimization. We report a CD7-targeted rAAV vector (CD7-AAV6/9) featuring a nanobody-fused hybrid capsid derived from a rationally selected chimeric combination of AAV6 and AAV9. CD7-AAV6/9 enables efficient and selective transduction of immortalized and primary human T and NK cells in vitro and in vivo in a humanized mouse model, achieves high production titers, and exhibits markedly reduced off-target transduction compared with wild-type serotypes. In parallel, we demonstrate that incorporation of a human gene-derived intron into the vector genome overcomes host-mediated transcriptional repression and enables robust transgene expression in human CD7 T lymphocyte and NK cell populations. To our knowledge, this represents the first application of intron-mediated enhancement in a rAAV vector context. Together, our findings establish an integrated capsid-genome design framework for targeting human T and NK cells, notoriously challenging immune cell populations for gene therapy, and provide a versatile platform readily adaptable to alternative surface markers and therapeutic payloads.