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.

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.

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.

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.

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.

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.

Identification and Validation of Small Molecules That Enhance Recombinant Adeno-associated Virus Transduction following High-Throughput Screens.

UNLABELLED: While the recent success of adeno-associated virus (AAV)-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2,396 compounds. We identified 13 compounds that were capable of enhancing transduction, of which 12 demonstrated vector-specific effects and 1 could also enhance vector-independent transgene expression. Many of these compounds had similar properties and could be categorized into five groups: epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors. In vivo analysis of rAAV2 and all of the clinically relevant compounds revealed that, consistent with our in vitro results, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectors in vivo using an AAV8 capsid that is known to exhibit robust liver tropism. Consistent with our in vitro results, teniposide coadministration greatly enhanced fragmented rAAV8 transduction at 48 h and 8 days. This study provides a foundation based on the rAAV small-molecule screen methodology, which is ideally used for more-diverse libraries of compounds that can be tested for potentiating rAAV transduction. IMPORTANCE: This study seeks to enhance the capability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs were tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50 to 100 times better with than without teniposide, a compound that is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy, which could result in safe and effective treatments for diverse acquired or genetic diseases.

Low-field thermal mixing in [1-(13)C] pyruvic acid for brute-force hyperpolarization.

We detail the process of low-field thermal mixing (LFTM) between (1)H and (13)C nuclei in neat [1-(13)C] pyruvic acid at cryogenic temperatures (4-15 K). Using fast-field-cycling NMR, (1)H nuclei in the molecule were polarized at modest high field (2 T) and then equilibrated with (13)C nuclei by fast cycling (∼300-400 ms) to a low field (0-300 G) that activates thermal mixing. The (13)C NMR spectrum was recorded after fast cycling back to 2 T. The (13)C signal derives from (1)H polarization via LFTM, in which the polarized ('cold') proton bath contacts the unpolarised ('hot') (13)C bath at a field so low that Zeeman and dipolar interactions are similar-sized and fluctuations in the latter drive (1)H-(13)C equilibration. By varying mixing time (tmix) and field (Bmix), we determined field-dependent rates of polarization transfer (1/τ) and decay (1/T1m) during mixing. This defines conditions for effective mixing, as utilized in 'brute-force' hyperpolarization of low-γ nuclei like (13)C using Boltzmann polarization from nearby protons. For neat pyruvic acid, near-optimum mixing occurs for tmix∼ 100-300 ms and Bmix∼ 30-60 G. Three forms of frozen neat pyruvic acid were tested: two glassy samples, (one well-deoxygenated, the other O2-exposed) and one sample pre-treated by annealing (also well-deoxygenated). Both annealing and the presence of O2 are known to dramatically alter high-field longitudinal relaxation (T1) of (1)H and (13)C (up to 10(2)-10(3)-fold effects). Here, we found smaller, but still critical factors of ∼(2-5)× on both τ and T1m. Annealed, well-deoxygenated samples exhibit the longest time constants, e.g., τ∼ 30-70 ms and T1m∼ 1-20 s, each growing vs. Bmix. Mixing 'turns off' for Bmix > ∼100 G. That T1m≫τ is consistent with earlier success with polarization transfer from (1)H to (13)C by LFTM.

Correction: Low-field thermal mixing in [1-(13)C] pyruvic acid for brute-force hyperpolarization.

Correction for 'Low-field thermal mixing in [1-(13)C] pyruvic acid for brute-force hyperpolarization' by David T. Peat et al., Phys. Chem. Chem. Phys., 2016, 18, 19173-19182.

Brute-Force Hyperpolarization for NMR and MRI.

Hyperpolarization (HP) of nuclear spins is critical for ultrasensitive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). We demonstrate an approach for >1500-fold enhancement of key small-molecule metabolites: 1-(13)C-pyruvic acid, 1-(13)C-sodium lactate, and 1-(13)C-acetic acid. The (13)C solution NMR signal of pyruvic acid was enhanced 1600-fold at B = 1 T and 40 °C by pre-polarizing at 14 T and ∼2.3 K. This "brute-force" approach uses only field and temperature to generate HP. The noted 1 T observation field is appropriate for benchtop NMR and near the typical 1.5 T of MRI, whereas high-field observation scales enhancement as 1/B. Our brute-force process ejects the frozen, solid sample from the low-T, high-B polarizer, passing it through low field (B < 100 G) to facilitate "thermal mixing". That equilibrates (1)H and (13)C in hundreds of milliseconds, providing (13)C HP from (1)H Boltzmann polarization attained at high B/T. The ejected sample arrives at a room-temperature, permanent magnet array, where rapid dissolution with 40 °C water yields HP solute. Transfer to a 1 T NMR system yields (13)C signals with enhancements at 80% of ideal for noted polarizing conditions. High-resolution NMR of the same product at 9.4 T had consistent enhancement plus resolution of (13)C shifts and J-couplings for pyruvic acid and its hydrate. Comparable HP was achieved with frozen aqueous lactate, plus notable enhancement of acetic acid, demonstrating broader applicability for small-molecule NMR and metabolic MRI. Brute-force avoids co-solvated free-radicals and microwaves that are essential to competing methods. Here, unadulterated samples obviate concerns about downstream purity and also exhibit slow solid-state spin relaxation, favorable for transporting HP samples.

Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication.

Tripartite motif proteins are important viral restriction factors and affect processes ranging from uncoating to transcription to immune signaling. Specifically, the promyelocytic leukemia protein (TRIM19; also called PML) is a viral restriction factor inhibiting processes from uncoating to transcription to cell survival. Here we investigated PML's effect on adeno-associated virus (AAV), a parvovirus used for gene delivery. Although dependovirus (AAV) and autonomous parvovirus (minute virus of mice) replication centers can colocalize with PML, PML's functional effect on parvoviruses is unknown. Using PML knockout mice, we determined that PML knockout enhances recombinant AAV2 (rAAV2) transduction at a range of vector doses in both male and female mice. In fact, male and female PML knockout mice exhibited up to 56-fold and 28-fold increases in transduction, respectively. PML inhibited several rAAV serotypes, suggesting a conserved mechanism, and organ specificity correlated with PML expression. Mechanistically, PML inhibited rAAV second-strand DNA synthesis, precluding inhibition of self-complementary rAAV, and did not affect the prior steps in transduction. Furthermore, we confirmed the effect of human PML on rAAV transduction through small interfering RNA (siRNA)-mediated knockdown in HuH7 cells and determined that the highest level of inhibition was due to effects of PML isoform II (PMLII). Overexpression of PMLII resulted in inhibition of second-strand synthesis, vector production, and genome replication. Moreover, wild-type AAV2 production and infectivity were also inhibited by PMLII, demonstrating a PML interaction with wild-type AAV. These data have important implications for AAV-mediated gene therapy. Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses.

Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway.

A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles.

Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration.

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

BACKGROUND: The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. METHODS: Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . RESULTS: We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. CONCLUSIONS: In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

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.

The prevalence of human parvovirus B19 DNA and antibodies in blood donors from four Chinese blood centers.

BACKGROUND: Human parvovirus B19 is a common human pathogen that causes a variety of diseases with outcomes ranging from asymptomatic to severe, especially in immunocompromised patients. The B19 virus can be transmitted via blood and/or blood products and its resistance to common viral inactivation and/or removal methods raises the importance of B19-related blood safety. However, the existence, variation, and loading of B19 in Chinese blood donors have not been determined. STUDY DESIGN AND METHODS: Quantitative polymerase chain reaction (PCR) was developed to detect all three genotypes of the human erythrovirus DNA in plasma samples. In total, 3957 donations from four Chinese blood centers were screened for B19 by real-time minipool nucleic acid amplification technology (NAT). The positive samples were then confirmed by nested PCR and subjected to sequence analysis and alignment for phylogenetic studies. An enzyme-linked immunosorbent assay-based experiment was also performed to identify the prevalence of immunoglobulin (Ig)G and/or IgM antibodies specific to the B19 structural proteins in acquired samples. RESULTS: Of 3957 blood donors, 23 (0.58%) specimens were found positive for B19 DNA. The quantitative DNA levels ranged from 2.48 × 10(2) to 6.38 × 10(4) copies/mL. The phylogenic analyses showed that the prevalent genotypes in Chinese blood donors belong to B19 Genotype 1. A total of 448 samples from Chinese blood donors were investigated for the seroprevalence of B19 antibodies, among which 24.6 and 6.9% of specimens were seropositive for B19 IgG and IgM antibodies, respectively. A total of 2.5% of these samples were positive for both antibody isotypes. CONCLUSIONS: Whether B19 NAT screening of blood and blood products should be launched in China, larger studies are needed to facilitate an informed decision.

Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application.

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

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 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.