AAV-mediated expression of HLA-G for the prevention of experimental ocular graft vs. host disease.

Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD.

Inhibition of experimental autoimmune uveitis by intravitreal AAV-Equine-IL10 gene therapy.

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.

Therapeutic Applications of Adeno-Associated Virus (AAV) Gene Transfer of HLA-G in the Eye.

The purpose of this paper is to review human leukocyte antigen G (HLA-G) in the eye, its role in immune tolerance, and the potential therapeutic use of AAV gene transfer and expression of HLA-G in various ocular tissues. Several studies are reviewed that demonstrate efficacy in animal models of disease, including intracorneal delivery of AAV-HLA-G to treat corneal inflammation and prevent corneal graft rejection, subconjunctival injection of AAV-HLA-G for ocular graft vs. host disease and potentially dry eye disease, and intravitreal injection of AAV-HLA-G to inhibit uveitis. Furthermore, due to the anti-vascular function of HLA-G, AAV-HLA-G may be an effective therapy for posterior ocular diseases, such as neovascular age-related macular degeneration, diabetic retinopathy, and choroidal neovascularization. Therefore, AAV-mediated gene transfer of HLA-G may be an effective treatment for common immune-mediated, inflammatory, and neovascular diseases of the eye.

Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy.

Adeno-associated virus (AAV) was first characterized as small "defective" contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.

Adeno-Associated Virus Mediated Gene Therapy for Corneal Diseases.

According to the World Health Organization, corneal diseases are the fourth leading cause of blindness worldwide accounting for 5.1% of all ocular deficiencies. Current therapies for corneal diseases, which include eye drops, oral medications, corrective surgeries, and corneal transplantation are largely inadequate, have undesirable side effects including blindness, and can require life-long applications. Adeno-associated virus (AAV) mediated gene therapy is an optimistic strategy that involves the delivery of genetic material to target human diseases through gene augmentation, gene deletion, and/or gene editing. With two therapies already approved by the United States Food and Drug Administration and 200 ongoing clinical trials, recombinant AAV (rAAV) has emerged as the in vivo viral vector-of-choice to deliver genetic material to target human diseases. Likewise, the relative ease of applications through targeted delivery and its compartmental nature makes the cornea an enticing tissue for AAV mediated gene therapy applications. This current review seeks to summarize the development of AAV gene therapy, highlight preclinical efficacy studies, and discuss potential applications and challenges of this technology for targeting corneal diseases.

Adeno-Associated Virus Vector Mobilization, Risk Versus Reality.

Recombinant adeno-associated viral (rAAV) vector 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 helper virus-independent (vector alone) or helper virus-dependent (de novo rAAV production facilitated by superinfection of both wild-type AAV [wtAAV] and Adenovirus 5 [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-Nano-dysferlin (4.9 kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and an 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 defined. 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 Ad-independent mobilization). In rAAV-infected cells coinfected with Ad and wtAAV, rAAV particle production was increased >50-fold compared with no Ad conditions. In addition, Ad-dependent rAAV vectors mobilized and resulted in >1,000-fold transduction upon a subsequent second-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.

Ocular Tolerability and Immune Response to Corneal Intrastromal AAV-IDUA Gene Therapy in New Zealand White Rabbits.

The chronic ocular toxicity, tolerability, and inflammation following corneal intrastromal injection of saline or escalating doses of an adeno-associated virus (AAV) containing a codon-optimized α-l-iduronidase (AAV-opt-IDUA) expression cassette were evaluated in New Zealand White rabbits. Corneal opacity following corneal intrastromal injection resolved by 24 h. Mild elevation of clinical ocular inflammation was observed 24 h after injection, but it returned to baseline by day 7 and no abnormalities were noted through 6 months of observation after injection. Vector genomes and IDUA cDNA were detected in the injected corneas in a dose-dependent manner. Both the lowest administered AAV-opt-IDUA dose, shown to be effective in mucopolysaccharidosis type I (MPS I) dogs, and a 10-fold higher dose of AAV-opt-IDUA resulted in no detectable immunologic response or adverse effect in rabbits. Vector genomes outside of the eye were rarely detected following corneal intrastromal injection of AAV-opt-IDUA, and neutralizing antibodies to the AAV capsid were not present at the experimental conclusion. This study, combined with our previous studies in MPS I dogs, suggests that AAV-opt-IDUA corneal gene therapy following corneal intrastromal injection of AAV-opt-IDUA has the potential to prevent and reverse blindness in MPS I patients in a safe and effective manner.

Preparation and Administration of Adeno-associated Virus Vectors for Corneal Gene Delivery.

Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.

Intrastromal Gene Therapy Prevents and Reverses Advanced Corneal Clouding in a Canine Model of Mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.

Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression.

Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated virus (AAV) genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat (ITR) sequences. These cis-elements, primarily from AAV serotype 2, are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution; however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1-4, 6, and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression both in vitro and in vivo. However, we observed three classes of AAV ITR expression in vitro. Class I ITRs (AAV2 and 3) generated the highest level, whereas class II (AAV 4) had intermediate levels, and class III (AAV1 and 6) had the lowest levels. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. In vivo, all classes had promoter activity. Next-generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the Rep binding element. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influence on transgene expression cassettes independent of promoter selection.

AAV-mediated expression of HLA-G1/5 reduces severity of experimental autoimmune uveitis.

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.

Gene Delivery to Human Limbal Stem Cells Using Viral Vectors.

Limbal stem cell (LSC) transplantation is a promising treatment for ocular surface diseases especially LSC deficiency. Genetic engineering represents an attractive strategy to increase the potential for success in LSC transplantations either by correcting autologous diseased LSCs or by decreasing the immunogenicity of allogeneic LSCs. Therefore, two popular viral vectors, adeno-associated viral (AAV) vector and lentiviral (LV) vector, were compared for gene delivery in human LSCs. Transduction efficiency was evaluated by flow cytometry, quantitation of viral genomes, and fluorescence microscopy after introducing eight self-complementary AAV serotypes or LV carrying a green fluorescent protein (GFP) cassette to fresh limbal epithelial cells, cultivated LSC colonies, or after corneal intrastromal injection into human explant tissue. For fresh limbal epithelial cells, AAV6 showed the highest transduction efficiency, followed by LV and AAV4 at 24 h after vector incubation, which did not directly correlate with internalized genome copy number. The colony formation efficiency, as well as colony size over time, showed no significant differences among AAV serotypes, LV, and nontreated controls. The percentage of GFP+ colonies at 14 days post-seeding was significantly higher in the LV group, which plateaued at 50% GFP+ upon serial passages. Interestingly, AAV6-treated colonies initially showed a variegated transduction phenotype with no GFP+ colonies in serial passages. Quantitative polymerase chain reaction and AAV6 capsid staining revealed that transduction was restricted to differentiated cells of LSC colonies at a post-entry step. Following central intrastromal injection of human corneas, both LV and AAV6 transduced the stroma and endothelial cells, and AAV6 also transduced cells of the epithelia. However, no transduction was observed in derived LSC colonies. The collective results demonstrate the effectiveness of LV for stable human LSC genetic engineering and an unreported phenomenon of AAV6 transduction restriction in multipotent cells derived from the human limbus.

Serotype survey of AAV gene delivery via subconjunctival injection in mice.

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction.

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-ß expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Inhibition of antigen presentation during AAV gene therapy using virus peptides.

The clinical trial using adeno-associated virus (AAV) vector delivery of mini-dystrophin in patients with Duchenne Muscular Dystrophy (DMD) demonstrated a cytotoxic lymphocyte (CTL) response targeting the transgene product. These mini-dystrophin-specific T-cells have the potential to clear all transduced muscle, presenting the general gene therapy concern of overcoming the CTL response to foreign proteins that provide therapeutic benefit. In this study, we exploited a natural immunosuppression strategy employed by some viruses that results in CTL evasion only in transduced cells. After transfection of the plasmids encoding viral peptides and ovalbumin, which includes the immune-domain epitope SIINFEKL, several viral small peptides (ICP47 and US6) inhibited the SIINFEKL peptide presentation. A single AAV vector genome that consisted of either transgene AAT fused with SIINFEKL epitope and, separately, ICP47 expressed from different promoters or a single fusion protein with ICP47 linked by a furin cleavage peptide (AATOVA-ICP47) decreased antigen presentation. Compared with AAV/AATOVA in which decreased AAT expression was observed at late time points, persistent transgene expression was obtained after systemic administration of AAV/AATOVA-ICP47 vectors in mice. We extended this strategy to DMD gene therapy. After administration of AAV vector encoding human mini-dystrophin fusion protein with ICP47 into mdx mice, a lower mini-dystrophin-specific CTL response was induced. Importantly, the ICP47 fusion to mini-dystrophin inhibited CTLs mediated cytotoxicity. Although demonstrated herein using AAT and mini-dystrophin transgenes in an AAV context, the collective results have implications for all gene therapy applications resulting in foreign peptides by immune suppression in only genetically modified cells.

AAV vector-meditated expression of HLA-G reduces injury-induced corneal vascularization, immune cell infiltration, and fibrosis.

Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated. Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLA-G5 (soluble) isoforms were validated in vitro. Then, following a corneal intrastromal injection, AAV vector transduction kinetics, using a chimeric AAV capsid, were determined in rabbits. One week following corneal trauma, a single intrastromal injection of scAAV8G9-optHLA-G1 + G5 prevented corneal vascularization, inhibited trauma-induced T-lymphocyte infiltration (some of which were CD8+), and dramatically reduced myofibroblast formation compared to control treated eyes. Biodistribution analyses suggested AAV vectors persisted only in the trauma-induced corneas; however, a neutralizing antibody response to the vector capsid was observed inconsistently. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to safely and effectively treat corneal vascularization and inhibit fibrosis while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

Structure-Based Designed Nano-Dysferlin Significantly Improves Dysferlinopathy in BLA/J Mice.

Dysferlinopathy is an autosomal recessive muscular dystrophy characterized by the progressive loss of motility that is caused by mutations throughout the DYSF gene. There are currently no approved therapies that ameliorate or reverse dysferlinopathy. Gene delivery using adeno-associated vectors (AAVs) is a leading therapeutic strategy for genetic diseases; however, the large size of dysferlin cDNA (6.2 kB) precludes packaging into a single AAV capsid. Therefore, using 3D structural modeling and hypothesizing dysferlin C2 domain redundancy, a 30% smaller, dysferlin-like molecule amenable to single AAV vector packaging was engineered (termed Nano-Dysferlin). The intracellular distribution of Nano-Dysferlin was similar to wild-type dysferlin and neither demonstrated toxicity when overexpressed in dysferlin-deficient patient myoblasts. Intramuscular injection of AAV-Nano-Dysferlin in young dysferlin-deficient mice significantly improved muscle integrity and decreased muscle turnover 3 weeks after treatment, as determined by Evans blue dye uptake and central nucleated fibers, respectively. Systemically administered AAV-Nano-Dysferlin to young adult dysferlin-deficient mice restored motor function and improved muscle integrity nearly 8 months after a single injection. These preclinical data are the first report of a smaller dysferlin variant tailored for AAV single particle delivery that restores motor function and, therefore, represents an attractive candidate for the treatment of dysferlinopathy.

Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.

Hip region muscular dystrophy and emergence of motor deficits in dysferlin-deficient Bla/J mice.

The identification of a dysferlin-deficient animal model that accurately displays both the physiological and behavior aspects of human dysferlinopathy is critical for the evaluation of potential therapeutics. Disease progression in dysferlin-deficient mice is relatively mild, compared to the debilitating human disease which manifests in impairment of particular motor functions. Since there are no other known models of dysferlinopathy in other species, locomotor proficiency and muscular anatomy through MRI (both lower leg and hip region) were evaluated in dysferlin-deficient B6.A-Dysfprmd /GeneJ (Bla/J) mice to define disease parameters for therapeutic assessment. Despite the early and progressive gluteal muscle dystrophy and significant fatty acid accumulation, the emergence of significant motor function deficits was apparent at approximately 1 year of age for standard motor challenges including the rotarod, a marble bury test, grip strength, and swimming speed. Earlier observations of decreased performance for Bla/J mice were evident during extended monitoring of overall exploration and rearing activity. Comprehensive treadmill gait analyses of the Bla/J model indicated significant differences in paw placement angles and stance in relation to speed and platform slope. At 18 months of age, there was no significant difference in the life expectancy of Bla/J mice compared to wild type. Consistent with progressive volume loss and fatty acid accumulation in the hip region observed by MRI, mass measurement of individual muscles confirmed gluteal and psoas muscles were the only muscles demonstrating a significant decrease in muscle mass, which is analogous to hip-girdle weakness observed in human dysferlin-deficient patients. Collectively, this longitudinal analysis identifies consistent disease parameters that can be indicators of efficacy in studies developing treatments for human dysferlin deficiency.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.