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Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of diseases. There are active clinical trials approaching a variety of inherited and acquired disorders of different organ systems. Approaches include ex vivo modification of hematologic stem cells (HSC), T lymphocytes and other immune cells, as well as in vivo delivery of genes or gene editing reagents to the relevant target cells by either local or systemic administration. In this article, we highlight success and ongoing challenges in three areas of high activity in gene therapy: inherited blood cell diseases by targeting hematopoietic stem cells, malignant disorders using immune effector cells genetically modified with chimeric antigen receptors, and ophthalmologic, neurologic, and coagulation disorders using in vivo administration of adeno-associated virus (AAV) vectors. In recent years, there have been true cures for many of these diseases, with sustained clinical benefit that exceed those from other medical approaches. Each of these treatments faces ongoing challenges, namely their high one-time costs and the complexity of manufacturing the therapeutic agents, which are biological viruses and cell products, at pharmacologic standards of quality and consistency. New models of reimbursement are needed to make these innovative treatments widely available to patients in need.

Recombinant adeno-associated virus (rAAV) has been widely used for gene delivery in animal models and successfully applied in clinical trials for treating inherited retinal disease. Although subretinal delivery of AAVs can effectively transduce photoreceptors and/or retinal pigmental epithelium (RPE), cells most affected by inherited retinal diseases, the procedure is invasive and complicated, and only delivers the gene to a limited retinal area. AAVs can also be delivered intravitreally to the retina, a much less invasive nonsurgical procedure. However, intravitreal administration of non-modified AAV serotypes tends to transduce only ganglion cells and inner nuclear layer cells. To date, most non-modified AAV serotypes that have been identified are incapable of efficiently transducing photoreceptors and/or RPE when delivered intravitreally. In this study, we investigate the retinal tropism of AAVrh10 vector administered by intravitreal injection to mouse, rat, and rabbit eyes. Our results demonstrate that AAVrh10 is capable of transducing not only inner retinal cells, but also outer retinal cells in all three species, though the transduction efficiency in rabbit was low. In addition, AAVrh10 preferentially transduced outer retinal cells in mouse models of retinal disease. Therefore, AAVrh10 vector could be a useful candidate to intravitreally deliver genes to photoreceptor and RPE cells.

Neurofibromatosis type 1, including the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), is featured by the loss of functional neurofibromin 1 (NF1) protein resulting from genetic alterations. A major function of NF1 is suppressing Ras activities, which is conveyed by an intrinsic GTPase-activating protein-related domain (GRD). In this study, we explored the feasibility of restoring Ras GTPase via exogenous expression of various GRD constructs, via gene delivery using a panel of adeno-associated virus (AAV) vectors in MPNST and human Schwann cells (HSCs). We demonstrated that several AAV serotypes achieved favorable transduction efficacies in those cells and a membrane-targeting GRD fused with an H-Ras C-terminal motif (C10) dramatically inhibited the Ras pathway and MPNST cells in a NF1-specific manner. Our results opened up a venue of gene replacement therapy in NF1-related tumors.

Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single- long terminal repeat (LTR) to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3'SIN-LTR's and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High-titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors.

Brain-derived neurotrophic factor (BDNF) gene delivery to the entorhinal cortex is a candidate for treatment of Alzheimer's disease (AD) to reduce neurodegeneration that is associated with memory loss. Accurate targeting of the entorhinal cortex in AD is complex due to the deep and atrophic state of this brain region. Using MRI-guided methods with convection-enhanced delivery, we were able to accurately and consistently target AAV2-BDNF delivery to the entorhinal cortex of non-human primates; 86 ± 3% of transduced cells in the targeted regions co-localized with the neuronal marker NeuN. The volume of AAV2-BDNF (3 × 108 vg/µl) infusion linearly correlated with the number of BDNF labeled cells and the volume (mm3) of BDNF immunoreactivity in the entorhinal cortex. BDNF is normally trafficked to the hippocampus from the entorhinal cortex; in these experiments, we also found that BDNF immunoreactivity was elevated in the hippocampus following therapeutic BDNF vector delivery to the entorhinal cortex, achieving growth factor distribution through key memory circuits. These findings indicate that MRI-guided infusion of AAV2-BDNF to the entorhinal cortex of the non-human primate results in safe and accurate targeting and distribution of BDNF to both the entorhinal cortex and the hippocampus. These methods are adaptable to human clinical trials.

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery-intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with AAV5 viral vector after parenchymal delivery. AAV5-CAG-GFP (1 × 1013 vector genomes per milliliter (vg ml-1)) was bilaterally infused either into putamen, thalamus or with the combination left putamen and right thalamus. Robust expression of GFP was seen throughout infusion sites and also in other distal nuclei. Interestingly, thalamic infusion of AAV5 resulted in the transduction of the entire corticospinal axis, indicating transport of AAV5 over long distances. Regardless of site of injection, AAV5 transduced both neurons and astrocytes equally. Our data demonstrate that AAV5 is a very powerful vector for the central nervous system and has potential for treatment of a wide range of neurological pathologies with cortical, subcortical and/or spinal cord affection.

Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone's ability to cross the blood-brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders.

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may have an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of the isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that (1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, (2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, (3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression and (4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain.

Adeno-associated virus serotype 1 (AAV1) has many advantages as a gene therapy vector, but the presence of pre-existing neutralizing antibodies (NAbs) is an important limitation. This study was designed to determine: (1) characteristics of AAV NAbs in human subjects, (2) prevalence of AAV1 NAbs in heart failure patients and (3) utility of aggressive immunosuppressive therapy in reducing NAb seroconversion in an animal model. NAb titers were assessed in a cohort of heart failure patients and in patients screened for a clinical trial of gene therapy with AAV1 carrying the sarcoplasmic reticulum calcium ATPase gene (AAV1/SERCA2a). AAV1 NAbs were found in 59.5% of 1552 heart failure patients. NAb prevalence increased with age (P=0.001) and varied geographically. The pattern of NAb titers suggested that exposure is against AAV2, with AAV1 NAb seropositivity due to crossreactivity. The effects of immunosuppression on NAb formation were tested in mini-pigs treated with immunosuppressant therapy before, during and after a single AAV1/SERCA2a infusion. Aggressive immunosuppression did not prevent formation of AAV1 NAbs. We conclude that immunosuppression is unlikely to be a viable solution for repeat AAV1 dosing. Strategies to reduce NAbs in heart failure patients are needed to increase eligibility for gene transfer using AAV vectors.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated virus (AAV) administration to newborn monkeys. Serotype rh10 AAV expressing ovalbumin and green fluorescent protein (GFP) was administered intravenously at birth and compared with vehicle controls. At 4 months postnatal age, a second injection was administered intramuscularly, followed by vaccination at 1 year of age with ovalbumin and GFP. Ovalbumin was highest 2 weeks post administration in the treated monkey, which declined but remained detectable thereafter; controls demonstrated no expression. Long-term AAV genome copies were present in myocytes. At 4 weeks, neutralizing antibodies to rh10 were present in the experimental animal only. With AAV9 administration at 4 months, controls showed transient ovalbumin expression that disappeared with the development of strong anti-ovalbumin and anti-GFP antibodies. In contrast, increased and maintained ovalbumin expression was noted in the monkey administered AAV at birth, without antibody development. After vaccination, the experimental monkey maintained levels of ovalbumin without antibodies, whereas controls demonstrated high levels of antibodies. These preliminary studies suggest that newborn AAV administration expressing secreted and intracellular xenogenic proteins may result in persistent expression in muscle, and subsequent vector administration can result in augmented expression without humoral immune responses.

The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, for example, age-related macular degeneration. We hypothesized that adeno-associated viral vector (AAV)-mediated sFLT1 expression could be used to inhibit abnormal brain angiogenesis. We tested the anti-angiogenic effect of sFLT1 and the feasibility of using AAV serotype 9 to deliver sFLT1 through intravenous injection (IV) to the brain angiogenic region. AAVs were packaged in AAV serotypes 1 and 2 (stereotactic injection) and 9 (IV injection). Brain angiogenesis was induced in adult mice through stereotactic injection of AAV1-VEGF. AAV2-sFLT02 containing sFLT1 VEGF-binding domain (domain 2) was injected into the brain angiogenic region, and AAV9-sFLT1 was injected into the jugular vein at the time of or 4 weeks after AAV1-VEGF injection. We showed that AAV2-sFLT02 inhibited brain angiogenesis at both time points. IV injection of AAV9-sFLT1 inhibited angiogenesis only when the vector was injected 4 weeks after angiogenic induction. Neither lymphocyte infiltration nor neuron loss was observed in AAV9-sFLT1-treated mice. Our data show that systemically delivered AAV9-sFLT1 inhibits angiogenesis in the mouse brain, which could be utilized to treat brain angiogenic diseases such as brain arteriovenous malformation.

Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.

Effective gene therapy strategies for the treatment of kidney disorders remain elusive. We report an optimized kidney-targeted gene delivery strategy using recombinant adeno-associated virus (rAAV) administered via retrograde renal vein injection in mice. Renal vein injection of rAAV consistently resulted in superior kidney transduction compared with tail vein injection using as little as half the tail vein dose. We compared rAAV5, 6, 8 and 9, containing either green fluorescent protein (GFP) or luciferase reporter genes driven by the Cytomegalovirus promoter. We demonstrated that although rAAV6 and 8 injected via renal vein transduced the kidney, transgene expression was mainly restricted to the medulla. Transgene expression was systematically low after rAAV5 injection, attributed to T-cell immune response, which could be overcome by transient immunosuppression. However, rAAV9 was the only serotype that permitted high-transduction efficiency of both the cortex and medulla. Moreover, both the glomeruli and tubules were targeted, with a higher efficiency within the glomeruli. To improve the specificity of kidney-targeted gene delivery with rAAV9, we used the parathyroid hormone receptor 'kidney-specific' promoter. We obtained a more efficient transgene expression within the kidney, and a significant reduction in other tissues. Our work represents the first comprehensive and clinically relevant study for kidney gene delivery.

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. On the basis of their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N and T368A mutations enhanced serum resistance, and additionally K66T, T368A and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy.

Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P

An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.

An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.

Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related increase in IL-1ra levels with a decrease in PGE 2 levels was observed, with the peak IL-1ra concentration being observed 7 days post-treatment in all groups. Perivascular infiltration with mononuclear cells was observed within the synovial membrane of the joint treated with the highest viral dose of 5 × 10 12 vg, but this was absent in the lower-dosed joints. The second-highest dose of scAAVeqIL-1ra 5 × 10 11 vg demonstrated elevated IL-1ra levels without any cellular response in the synovium. Taken together, the data suggest that the 10-fold lower dose of 5 × 10 11 vg scAAVIL-1ra would be a safe therapeutic dose in an equine model of PTOA., (© 2023. The Author(s).)

Adeno-associated virus (AAV) vector-based gene therapy is potentially curative for various genetic diseases; however, the development of a scalable purification method for full-genome AAV vectors remains crucial to increase productivity and reduce cost of GMP production. In this study, we developed a large-scale short-term purification method for functional full-genome AAV particles by using 2-step cesium chloride (CsCl) density-gradient ultracentrifugation with a zonal rotor. The 2-step CsCl method with a zonal rotor improves separation between empty and full-genome AAV particles, reducing the ultracentrifugation time (4-5 h) and increasing the AAV volume for purification. The highly purified full-genome AAV particles were confirmed by analytical ultracentrifugation (AUC), droplet digital PCR (ddPCR) in the whole region of the AAV vector genome, transduction efficiency in target cells, and transmission electronic microscopy (TEM). The high-purity AAV9 particles were obtained using culture supernatant during vector preparation rather than cell lysate. CsCl could be simply removed by a hydroxyapatite column. Interestingly, ddPCR analysis revealed that "empty" AAV particles contain small fragments of the inverted terminal repeat (ITR), probably due to unexpected packaging of Rep-mediated ITR fragments. This large-scale functional AAV vector purification with ultracentrifugation would be effective for gene therapy., (© 2023. The Author(s).)

Gene-based therapeutics are actively being pursued for the treatment of lung diseases. While promising advances have been made over the last decades, the absence of clinically available lung-directed genetic therapies highlights the difficulties associated with this effort. Largely, progress has been hindered by the presence of inherent physical and physiological airway barriers that significantly reduce the efficacy of gene transfer. These barriers include surface mucus, mucociliary action, cell-to-cell tight junctions, and the basolateral cell membrane location of viral receptors for many commonly used gene vectors. Accordingly, airway surface preparation methods have been developed to disrupt these barriers, creating a more conducive environment for gene uptake into the target airway cells. The two major approaches have been chemical and physical methods. Both have proven effective for increasing viral-mediated gene transfer pre-clinically, although with variable effect depending on the specific strategy employed. While such methods have been explored extensively in experimental settings, they have not been used clinically. This review covers the airway surface preparation strategies reported in the literature, the advantages and disadvantages of each method, as well as a discussion about applying this concept in the clinic., (© 2022. The Author(s).)

Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease., (© 2023. The Author(s).)

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)