Viral Vector-mediated Gene Transfer Research Articles

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Transduction of the Central Nervous System After Intracerebroventricular Injection of Adeno-Associated Viral Vectors in Neonatal and Juvenile Mice

Several neurodevelopmental and neurodegenerative disorders affecting the central nervous system are potentially treatable via viral vector-mediated gene transfer. Adeno-associated viral (AAV) vectors have been used in clinical trials because of their desirable properties including a high degree of safety, efficacy, and stability. Major factors affecting tropism, expression level, and cell type specificity of AAV-mediated transgenes include encapsidation of different AAV serotypes, promoter selection, and the timing of vector administration. In this study, we evaluated the ability of single-stranded AAV2 vectors pseudotyped with viral capsids from serotype 9 (AAV2/9) to transduce the brain and target gene expression to specific cell types after intracerebroventricular injection into mice. Titer-matched AAV2/9 vectors encoding the enhanced green fluorescent protein (eGFP) reporter, driven by the cytomegalovirus (CMV) promoter, or the neuron-specific synapsin-1 promoter, were injected bilaterally into the lateral ventricles of C57/BL6 mice on postnatal day 5 (neonatal) or 21 (juvenile). Brain sections were analyzed 25 days after injection, using immunocytochemistry and confocal microscopy. eGFP immunohistochemistry after neonatal and juvenile administration of viral vectors revealed transduction throughout the brain including the striatum, hippocampus, cerebral cortex, and cerebellum, but with different patterns of cell-specific gene expression. eGFP expression was seen in astrocytes after treatment on postnatal day 5 with vectors carrying the CMV promoter, expanding the usefulness of AAVs for modeling and treating diseases involving glial cell pathology. In contrast, injection of AAV2/9-CMV-eGFP on postnatal day 21 resulted in preferential transduction of neurons. Administration of AAV2/9-eGFP with the synapsin-1 promoter on either postnatal day 5 or 21 resulted in widespread neuronal transduction. These results outline efficient methods and tools for gene delivery to the nervous system by direct, early postnatal administration of AAV vectors. Our findings highlight the importance of promoter selection and age of administration on the intensity, distribution, and cell type specificity of AAV transduction in the brain.

BAFF Controls Neural Cell Survival through BAFF Receptor

Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell–activating factor (BAFF) and its receptor (BAFF-R) are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.

Mir‐142‐3p target sequences reduce transgene‐directed immunogenicity following intramuscular adeno‐associated virus 1 vector‐mediated gene delivery

Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood-related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen-presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences. In the present study, we explored the potential of mir-142-3p specific target sequences with respect to reducing or abolishing immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly. The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been described previously and resulted in the loss of OVA protein expression. In the present study, we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated into the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody levels. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing four or eight repeats of mir-142-3p target sequences after the OVA sequence were used. The rising humoral and cellular immune responses against OVA protein after intramuscular delivery can be efficiently reduced by the use of mir-142-3p target sequences.

Current progress on gene therapy for primary immunodeficiencies

Primary immunodeficiencies have played a major role in the development of gene therapy for monogenic diseases of the bone marrow. The last decade has seen convincing evidence of long-term disease correction as a result of ex vivo viral vector-mediated gene transfer into autologous haematopoietic stem cells. The success of these early studies has been balanced by the development of vector-related insertional mutagenic events. More recently the use of alternative vector designs with self-inactivating designs, which have an improved safety profile has led to the initiation of a wave of new studies that are showing early signs of efficacy. The ongoing development of safer vector platforms and gene-correction technologies together with improvements in cell-transduction techniques and optimised conditioning regimes is likely to make gene therapy amenable for a greater number of PIDs. If long-term efficacy and safety are shown, gene therapy will become a standard treatment option for specific forms of PID.

A Novel Strategy to Circumvent Pre-Existing Humoral Immunity to AAV

Abstract 2050Adeno-associated viral (AAV) vector-mediated gene transfer has shown great potential as a therapeutic platform for inherited and metabolic diseases. Systemic delivery of AAV vectors through the bloodstream is a safe, non-invasive, and potentially effective strategy to target a variety of organs, including liver, muscle, and brain. However, neutralizing antibodies (NAb) to AAV, highly prevalent in humans, constitute a major obstacle to successful gene transfer, particularly when a vector is delivered through the vasculature.Thus far, the liver was targeted to express the coagulation factor IX (F.IX) transgene in two clinical studies. In one study, a single-stranded AAV2 vector expressing the F.IX transgene was delivered through the hepatic artery to severe hemophilia B subjects at doses of 8×1010, 4×1011, and 2×1012 vector genomes (vg)/kg. Efficacy was observed in one subject from the high-dose cohort, who achieved peak F.IX transgene plasma levels of ∼10% of normal. The subjects infused at lower doses did not show any evidence of transgene expression, despite the fact that they did not have detectable NAb to AAV.In a second study, a self-complementary AAV8 vector expressing the F.IX transgene was delivered through peripheral vein infusion to severe hemophilia B subjects at doses similar to those administered in the AAV2 study, 2×1011, 6×1011, and 2×1012 vg/kg. All subjects enrolled in the AAV8 trial had evidence of transgene expression above baseline levels, despite the fact that some of the subjects had low-but-detectable anti-AAV8 NAb. Peak F.IX plasma levels at the high vector dose were 8–12% of normal, similar to the high dose of the AAV2 trial, suggesting that the vectors used in the two studies had comparable potency. Importantly, the vectors used in the two studies differed in empty capsid content, as the AAV2 vector preparation was essentially empty capsid-free and the AAV8 vector contained a 5–10 fold excess of empty capsids.The current study was undertaken to explore the role of empty capsids as a factor in the difference in outcome in the low- and mid- dose cohorts of the two trials. Our underlying hypothesis was that the presence of an excess of empty capsids effectively absorbs low-level neutralizing and non-neutralizing antibodies, and permits transduction even in their presence. Using a newly developed AAV antibody dot-blot assay, we demonstrate that adult human subjects with a low to undetectable NAb titer (1:1) as assessed by a commonly used assay do, in fact, carry significant amounts of anti-AAV antibodies. Conversely, children aged one year appear to be truly naïve for anti-AAV humoral immunity. Using C57BL/6 mice passively immunized with purified human IgG injected intraperitoneally 24 hours before vector administration, we further demonstrate that the same low levels of anti-AAV antibodies found in humans (NAb titer of 1:1–1:3) can block >90% of liver transduction after peripheral vein delivery of AAV8 vectors expressing F.IX at doses of 1×1012 vg/kg, comparable to those tested in the clinic. We next demonstrated that the inhibitory effect of low titer (1:1–1:3) anti-AAV antibodies can be overcome by adding a 5 to 10-fold excess of empty capsids to the final formulation of AAV8 vector, and that empty capsid content can be carefully titrated as a function of the animal’s anti-AAV NAb in order to achieve efficient target organ transduction, even at titers >1:100. However, the beneficial effect of empty capsids on liver transduction is lost when a 1000-fold excess of AAV8 empty capsids are added to the formulation of AAV8 vectors, due to receptor binding competition. This inhibitory effect could be avoided by using AAV2 empty capsids, which efficiently protect AAV8 vectors from NAb without inhibiting transduction. These results were confirmed in non-human primates, a natural host for AAV8, in which a 5 to 6-fold increase in liver transduction was achieved by formulating vector in 5–10 fold excess AAV8 empty capsids, reaching levels of F.IX expression of 10 to 20% of normal. Application of these findings to the development of personalized formulations of vector product for intravascular delivery will facilitate safe, effective AAV-mediated gene transfer in settings in which vectors are delivered through the systemic circulation. Disclosures:Mingozzi:Children’s Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Anguela:Children’s Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Wright:Children’s Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. High:Children’s Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties.

Vascular Endothelial Growth Factor–Mediated Islet Hypervascularization and Inflammation Contribute to Progressive Reduction of β-Cell Mass

Type 2 diabetes (T2D) results from insulin resistance and inadequate insulin secretion. Insulin resistance initially causes compensatory islet hyperplasia that progresses to islet disorganization and altered vascularization, inflammation, and, finally, decreased functional β-cell mass and hyperglycemia. The precise mechanism(s) underlying β-cell failure remain to be elucidated. In this study, we show that in insulin-resistant high-fat diet-fed mice, the enhanced islet vascularization and inflammation was parallel to an increased expression of vascular endothelial growth factor A (VEGF). To elucidate the role of VEGF in these processes, we have genetically engineered β-cells to overexpress VEGF (in transgenic mice or after adeno-associated viral vector-mediated gene transfer). We found that sustained increases in β-cell VEGF levels led to disorganized, hypervascularized, and fibrotic islets, progressive macrophage infiltration, and proinflammatory cytokine production, including tumor necrosis factor-α and interleukin-1β. This resulted in impaired insulin secretion, decreased β-cell mass, and hyperglycemia with age. These results indicate that sustained VEGF upregulation may participate in the initiation of a process leading to β-cell failure and further suggest that compensatory islet hyperplasia and hypervascularization may contribute to progressive inflammation and β-cell mass loss during T2D.

Advances in Gene Delivery Systems.

The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retro-virus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral- and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives.

Novel Therapies, High-Risk Pediatric Research, and the Prospect of Benefit: Learning from the Ethical Disagreements

Despite recent efforts by the US Food and Drug Administration (FDA) to improve the quality and quantity of clinical research data,1 two-thirds of drugs prescribed currently to children have not been studied for safety and efficacy in pediatric populations, and information on the efficacy and safety of drugs in children is not methodically collected and analyzed.2 According to some studies, the majority of pediatric drugs prescribed for children involve unlicensed drugs or off-label prescribing across all medication categories.3 There is evidence of a greater risk of a severe adverse drug reaction occurring in association with the off-label or unlicensed use of drugs in children.4

Effect of Nuclear Factor κB Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.

The endocannabinoid system: a key modulator of emotions and cognition

The endocannabinoid system: a key modulator of emotions and cognition

Adeno-Associated Viral Vector Mediated Gene Transfer for Hemophilia B

Abstract 5▪▪This icon denotes a clinically relevant abstract Background:Hemophilia B (HB), an X-linked bleeding disorder, is ideally suited for gene therapy. We investigated a novel approach using peripheral vein infusion of a single dose of a serotype-8 pseudotyped self-complementary adeno-associated virus (AAV) vector expressing a codon-optimized coagulation factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco). Methods:Six severe HB subjects (FIX ≤1%) were enrolled sequentially into one of three dose cohorts with two subjects in each group. Vector was administered without immunosuppression. The subjects were followed for 6–16 months post treatment. Results:AAV-mediated expression of FIX at 2–11% of normal was observed in all subjects. Four of the six have discontinued prophylaxis and remain free of spontaneous hemorrhage. The other two have increased the interval between FIX prophylaxes. A high-dose subject developed asymptomatic, transient elevation of serum transaminases associated with detection of AAV8 capsid specific T cells in peripheral blood. The second high-dose subject experienced a slight increase of liver enzymes, of less clear etiology. Treatment of each with a short course of steroids led to rapid normalization of the transaminases and maintenance of FIX levels in the 3–11% range. Conclusion:Peripheral vein administration of scAAV2/8-LP1-hFIXco was well tolerated and resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype. Immune-mediated clearance of AAV-transduced hepatocytes remains a concern but our data suggest that this process may be controlled with a short course of steroids without loss of transgene expression. Hence, our novel approach shows promise for gene therapy of HB and other protein deficiencies. (ClinicalTrials.gov number, NCT00979238) Disclosures:Nathwani:Amsterdam Molecular Therapeutics: Patents & Royalties. Gray:Amsterdam Molecular Therapeutics: Patents & Royalties. Davidoff:Amsterdam Molecular Therapeutics: Patents & Royalties.

Advances in Gene Delivery Systems

The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retrovirus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral- and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives.

A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100bp fragments that each support long-term expression, and are contained within an ~320bp fragment located ~3kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100bp fragments contained 44 or 23bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42bp sequence, centered on the 23bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16bp maximum essential sequence. This 16bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

Local and retrograde gene transfer into primate neuronal pathways via adeno-associated virus serotype 8 and 9

Viral vector-mediated gene transfer has become increasingly valuable for primate brain research, in particular for application of genetic methods (e.g. optogenetics) to study neuronal circuit functions. Neuronal cell tropisms and infection patterns are viable options for obtaining viral vector-mediated transgene delivery that is selective for particular neuronal pathways. For example, several types of viral vectors can infect axon terminals (retrograde infections), which enables targeted transgene delivery to neurons that directly project to a particular viral injection region. Although recent studies in rodents have demonstrated that adeno-associated virus serotype 8 (AAV8) and 9 (AAV9) efficiently transduce neurons, the tropisms and infection patterns remain poorly understood in primate brains. Here, we constructed recombinant AAV8 or AAV9, which expressed an enhanced green fluorescent protein (EGFP) gene driven by a ubiquitous promoter (AAV8-EGFP and AAV9-EGFP, respectively), and stereotaxically injected it into several brain regions in marmosets and macaque monkeys. Immunohistochemical analyses revealed almost exclusive colocalization of EGFP fluorescence via AAV9-mediated gene transfer with a neuron-specific marker, indicating endogenous neuronal tropism of AAV9, which was consistent with our previous results utilizing AAV8. Injections of either AAV8-EGFP or AAV9-EGFP into the marmoset striatum resulted in EGFP expression in local striatal neurons as a result of local infection, as well as expression in dopaminergic neurons of the substantia nigra via retrograde transport along nigrostriatal axonal projections. Retrograde infections were also observed in the frontal cortex and thalamus, which are known to have direct projections to the striatum. These local and retrograde gene transfers were further demonstrated in the geniculocortical pathway of the marmoset visual system. These findings indicate promising capabilities of AAV8 and AAV9 to deliver molecular tools into a range of primate neural systems in pathway-specific manners through their neuronal tropisms and infection patterns.

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is ∼145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18 × 10(8)-6.85 × 10(9) viral particles per fetus) was 24.6±0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways <100 μm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6±0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6

Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (~4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the ~14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (ΔH2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the ~1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 × 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.

AAV-Mediated Overexpression of the CB1 Receptor in the mPFC of Adult Rats Alters Cognitive Flexibility, Social Behavior, and Emotional Reactivity

The endocannabinoid (ECB) system is strongly involved in the regulation of cognitive processing and emotional behavior and evidence indicates that ECB signaling might affect these behavioral abilities by modulations of prefrontal cortical functions. The aim of the present study was to examine the role of the CB1 receptor in the medial prefrontal cortex (mPFC) on cognitive flexibility and emotional behavior. Therefore, the CB1 receptor was overexpressed by adeno-associated virus vector-mediated gene transfer specifically in the mPFC of adult Wistar rats. Animals were then tested in different anxiety-related paradigms for emotional reactivity [e.g., elevated plus maze (EPM), light/dark emergence test (EMT), social interaction] and the attentional set shift task (ASST) – an adaptation of the human Wisconsin card sorting test – for cognitive abilities and behavioral flexibility. A subtle increase in exploratory behavior was found in CB1 receptor overexpressing animals (CB1-R) compared to Empty vector injected controls (Empty) in the EMT and EPM, although general locomotor activity did not differ between the groups. During social interaction testing, social contact behavior toward the unknown conspecific was found to be decreased, whereas social withdrawal was increased in CB1-R animals and they showed an inadequate increase in exploratory behavior compared to control animals. In the ASST, impaired reversal learning abilities were detected in CB1-R animals compared to controls, indicating reduced behavioral flexibility. In conclusion, upregulation of the CB1 receptor specifically in the rat mPFC induces alterations in emotional reactivity, leads to inadequate social behavior, and impairs cognitive flexibility. These findings might be relevant for neuropsychiatric disorders, since higher cortical CB1 receptor expression levels as well as similar behavioral impairments as observed in the present study have been described in schizophrenic patients.

Emerging strategies for EphA2 receptor targeting for cancer therapeutics

Importance of the field: High mortality rates with cancers warrant further development of earlier diagnostics and better treatment strategies. Membrane-bound erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) is overexpressed in breast, prostate, urinary bladder, skin, lung, ovary and brain cancers.Areas covered in this review: EphA2 overexpression in cancers, its signaling mechanisms and strategies to target its deregulation.What the reader will gain: High EphA2 expression in cancer cells is correlated with a poor prognosis associated with recurrence due to enhanced metastasis. Interaction of the EphA2 receptor with its ligand (e.g., ephrinA1) triggers events that are deregulated and implicated in carcinogenesis. EphrinA1-independent oncogenic activity and ephrinA1-dependent tumor suppressor roles for EphA2 are described. Molecular interactions of EphA2 with signaling proteins are associated with the modulation of cytoskeleton dynamics, cell adhesion, proliferation, differentiation and metastasis. The deregulated signaling by EphA2 and its involvement in oncogenesis provide multiple avenues for the rational design of intervention approaches.Take home message: EphA2 has been tested as a drug target using multiple approaches such as agonist antibodies, RNA interference, immunotherapy, virus vector-mediated gene transfer, small-molecule inhibitors and nanoparticles. With over a decade of research, encouraging results with targeting of EphA2 expression in various pre-clinical cancer models necessitate further studies.

Phenotypic Correction of a Mouse Model of Hemophilia B by In Vivo Genetic Correction of the F9 Gene

AbstractAbstract LBA-5Inherited hematologic disorders have the potential to be effectively treated by gene therapy, with recent successes reported for several genetic disorders using viral vector-mediated gene transfer (ADA-SCID, NEJM 2009; β-thalassemia, Nature 2010). However, these trials and others illustrate some of the disadvantages and risks of using viral vector-based gene addition strategies, including loss of endogenous gene regulation and random insertion leading to potential for insertional mutagenesis. An alternative approach is gene correction, where in situ correction of a gene mutation allows endogenous gene regulation and decreases risks related to random integration. Gene correction is based on gene targeting, the therapeutic utility of which has historically been limited to mouse embryonic stem cells due to low homologous recombination rates in other cell types. However, a recently developed class of fusion proteins, zinc finger nucleases (ZFNs), have been shown to increase targeting efficiency 2–3 logs by inducing site-specific DNA double strand breaks at the intended targeting site. ZFNs have permitted high efficiency therapeutic gene targeting in a variety of cultured cells previously thought intractable to these processes, but ZFN-mediated gene correction has yet to be successfully achieved in vivo in an animal model of disease. Here we show ZFN-mediated therapeutic gene targeting of a mutated F9 gene in vivo, resulting in phenotypic correction of a mouse model of hemophilia B (HB). We first generated ZFNs targeting intron 1 of the human F9 gene (F9 ZFNs). We hypothesized the F9 ZFNs would mediate insertion of a wild-type F9 exons 2–8 minigene into intron 1 via gene targeting, thus bypassing the 95% of F9 mutations that occur in exons 2–8. We next generated a humanized HB mouse model with a deletion of the mouse F9 gene and knock-in (at the ROSA 26 locus) of a catalytic domain-deleted human F9 mini-gene (hF9mut) transgene. Adeno-associated viral (AAV) vector delivery of the F9 ZFNs to hF9mut mouse liver resulted in cleavage of the intron 1 target site in 45% of hepatocytes. We then generated an AAV donor vector containing a w.t. exons 2–8 insert flanked by arms of homology. Co-delivery of the AAV-ZFN and AAV-donor vectors to neonatal hF9mut mice (n=16) resulted in circulating F.IX levels of 120–350 ng/mL (2-7% of normal), whereas mice receiving AAV-ZFN alone (n=17) or AAV-mock & AAV-donor (n=15) had no detectable F.IX expression (detection limit 15 ng/mL), or <25 ng/mL F.IX, respectively. PCR analysis of liver DNA from ZFN+donor mice demonstrated genomic evidence of gene targeting at a rate of 2–7% of alleles. F.IX expression in ZFN+donor mice was shown to be stable after 5 months, with follow-up ongoing. In addition, there was no loss of expression following partial hepatectomy, which causes loss of expression from non-integrated episomes upon subsequent hepatocyte proliferation. F.IX expression was also shown to be specific, as opposed to resulting from random integration, as mice lacking the hF9mut gene averaged less than 30 ng/mL after receiving AAV-ZFN and AAV-donor. hF.IX RT-PCR on 10 different tissues confirmed liver-specific expression. To assess phenotypic correction, we performed aPTTs on mice that received ZFN+donor or mock+donor, as well as wild-type (WT) mice and HB mice. WT mice averaged 36 seconds, ZFN+donor mice averaged 44 seconds, mock+donor mice averaged 60 seconds, and HB mice averaged 67 seconds. There was no significant difference in aPTT between WT and ZFN+donor, or mock+donor and HB (p = 0.086 and 0.11, respectively). However, the aPTT for ZFN+donor mice was significantly shortened compared to mock+donor mice (p=0.0014), demonstrating phenotypic correction of the defect in clot formation in HB mice. To our knowledge this is the first demonstration of ZFN-driven gene correction in vivo, and the first demonstration of the in vivo use of ZFNs to correct an animal model of human disease. These results establish a novel paradigm for in vivo gene correction as a method for treating inherited hematologic diseases. Disclosures:Doyon:Sangamo Biosciences: Employment. Li:Sangamo Biosciences: Employment. Wong:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Rebar:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo: Employment. High:Sangamo Biosciences: Consultancy; Children's Hospital of Philadelphia: Patents & Royalties.