HD-Ad Vector Research Articles

In vitro and in vivo expansion of CD33/HBG promoter-edited HSPCs with Mylotarg

We developed an in vivo HSC gene therapy approach that consists of HSC mobilization and intravenous injection of HSC-tropic HDAd vectors. To achieve therapeutically relevant numbers of corrected cells, we incorporated in vivo expansion of transduced cells. We used an HDAd vector for a multiplex adenine base editing approach to i) remove the region within CD33 that is recognized by gemtuzumab ozogamicin (GO) (MylotargTM), and ii) create therapeutic edits within the HBG1/2 promoters to reactivate γ-globin/HbF. In vitro studies with HDAd-transduced human CD34+ cells showed editing of both targeted sites and a 2- to 3-fold GO-mediated expansion of edited erythroid/myeloid progenitors. After erythroid in vitro differentiation, up to 40% of erythrocytes were HbF positive. For in vivo studies, mice were transplanted with human CD34+ cells. After engraftment, HSCs were mobilized with G-CSF/AMD3100 followed by an intravenous HDAd injection and GO-mediated in vivo selection. Two months later, editing in human cells within the bone marrow was significantly higher in GO-treated mice. The percentage of HbF+ human erythroid cells was 2.5-fold greater compared to untreated mice. These data indicate that in vivo GO selection can increase edited erythroid cells.

HDAd6/35++ - A new helper-dependent adenovirus vector platform for in vivo transduction of hematopoietic stem cells

In previous studies, we achieved safe and efficient invivo hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34+ cells invitro. Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term invivo HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient invivo HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the invivo approach.

598. TARGTCNS for the Treatment of Central Nervous System Disorders

Diseases and disorders of the central nervous system (CNS) represent a large field of unmet need that significantly impacts the lifespan and quality of life of many patients. The ability to deliver therapeutic proteins from the systemic circulation to the CNS is hampered by the blood-brain barrier (BBB). A number of strategies have been developed to allow therapies to cross the BBB, but to date with limited success. We have developed a novel localized approach to deliver protein and peptide therapeutics to the CNS based on our proprietary Transduced Autologous Restorative Gene Therapy system (TARGT™) platform. TARGT is based on autologous dermal micro-organs (MO) harvested and transduced ex-vivo to produce a therapeutic protein and then re-implanted in the patient. In this study we assessed whether TARGT can survive and deliver sustained localized protein post implantation into the CNS. In rats, MOs, 2×1mm each, were harvested from Lewis rat skin, characterized in-vitro, and then implanted in the Lewis rats’ cisterna magna. The procedure was well-tolerated and without observed behavioral change in all implanted rats. Histopathology of MOs excised several weeks post implantation, demonstrated viable and integrated MOs without signs of implant rejection. In the second phase, rat MOs were processed ex-vivo into TARGT secreting erythropoietin (TARGTEPO) by transduction with Helper Dependent Adenoviral vector encoding human erythropoietin (HDAd-HuEPO). Rat TARGTs secreting human EPO were then implanted into Lewis rats’ cisterna magna. Post implantation, cerebrospinal fluid (CSF) and serum were collected and EPO levels were measured by HuEPO ELISA. Results obtained suggest that implantation was well tolerated with no observed signs of rejection. Measurable human EPO levels were detected in rat CSF for the duration of the experiment. Detectable but low levels of Hu-EPO were also measured in the rat serum, as expected, since CSF drains into the peripheral blood. In pigs we have tested the viability and monoclonal antibody secretion from TARGTs into the CNS. Humira, an established antibody drug, was selected as a proof of concept. Pig MOs were harvested and transduced with the HDAd vector carrying the Humira sequence. Two TARGTHumira were implanted in the pig subdural space at the parietal convexity region and the pig was followed for one week post implantation. Results obtained post implantation suggests no observed pig's behavioral change. Humira was measured in CSF samples taken from the implantation area and the lumbar space. One week post implantation, TARGTs were excised out of the pig brain when they are viable and no signs of inflammation or damage to the brain tissue were observed. These results demonstrate that TARGT is a promising novel therapeutic platform with the potential for treatment of CNS disorders. We are now actively studying TARGTCNS treatment of lysosomal storage diseases and brain malignancies.

105. A Single Administration of Low Dosage (5 x1010 vp/kg) hFX/HDAd Complex Achieves Saturated hFVIII Levels in the Blood of Mice

The development of helper-dependent adenoviral vectors (HDAds) devoid of all functional viral genes has largely overcome cellmediated host immune responses associated with Ad viral proteins. However, a dose-related acute toxicity associated with systemic delivery has been a major obstacle to the clinical translation of Adbased vectors. Since HDAd has a large transgene cargo (up to 34 kb), we recently succeeded in producing an HDAd encoding two codon-optimized hFVIII variant N6 expression cassettes (HDAd2xcoN6). We evaluated whether HDAd2xcoN6 improves hFVIII expression and activity in vivo. Hemophilia A (HA) mice were injected with a sub-lethal (human) dose (5 x1011 vp/kg) of HDAd2xcoN6 or HDAd1xcoN6 encoding a single expression cassette. HA mice injected with HDAd2xcoN6 exhibited over 10-fold higher hFVIII expression and activity compared to those injected with an equal titer of HDAd1xcoN6. Thus, we evaluated hFVIII expression after a systemic administration of 5 x1010 vp/kg (one log-lower than a human sub-lethal dose) of HDAd2xcoN6 in non-human primates. One primate showed 2% hFVIII levels in plasma compared to pretreatment. Though that expression was transient, it represents the first time that an Ad-based vector has shown a detectable level of therapeutic gene expression in the blood after systemic administration with such a low dosage in a large animal model. However, this result also suggests that the improvement of transduction of HDAd and transgene expression in the liver is required for long-lasting, high hFVIII expression in a large animal model.Previous reports have revealed that the adenoviral capsid interacts with coagulation factor X (FX) in the blood after systemic administration, and the interaction leads to transduction of this complex into the liver in conjunction with highly expressing HSPGs (receptor of FX/Ad5 complex). The complex of Ad5 with human FX (hFX/HDAd) has a greater affinity to HSPGs than mouse FX does. We therefore evaluated forming a complex of HDAd with hFX in order to improve HDAd liver transduction. Mice injected with 5 x1010 vp/kg of hFX/ HDAd2xcoN6 complex sustained 100% hFVIII levels in the blood until 4 weeks after injection. In contrast, mice injected with same dosage of HDAd2xcoN6 alone had undetectable levels of hFVIII in the blood 4 weeks post-injection, similar to our observations in non-human primate. Although mice injected with the hFX/HDAd complex showed 10-fold higher transgene products in vivo compared to mice treated with HDAd alone, HDAd vector copies in the livers of mice injected with the hFX/HDAd complex were only 2-fold higher. Interestingly, mice injected with the hFX/HDAd complex exhibited 10-fold higher transgene expression at mRNA level in the liver compared to mice injected with HDAd alone. These results suggest that the hFX/HDAd complex can lead not only to high liver transduction, but also to improving transgene expression at the transcriptional level after systemic administration. We are now investigating how the hFX/HDAd complex improves transgene expression at the transcriptional level.

Physiologic and metabolic safety of butyrylcholinesterase gene therapy in mice

In continuing efforts to develop gene transfer of human butyrylcholinesterase (BChE) as therapy for cocaine addiction, we conducted wide-ranging studies of physiological and metabolic safety. For that purpose, mice were given injections of adeno-associated virus (AAV) vector or helper-dependent adenoviral (hdAD) vector encoding human or mouse BChE mutated for optimal cocaine hydrolysis. Age-matched controls received saline or AAV-luciferase control vector. At times when transduced BChE was abundant, physiologic and metabolic parameters in conscious animals were evaluated by non-invasive Echo-MRI and an automated “Comprehensive Laboratory Animal Monitoring System” (CLAMS). Despite high vector doses (up to 1013 particles per mouse) and high levels of transgene protein in the plasma (∼1500-fold above baseline), the CLAMS apparatus revealed no adverse physiologic or metabolic effects. Likewise, body composition determined by Echo-MRI, and glucose tolerance remained normal. A CLAMS study of vector-treated mice given 40mg/kg cocaine showed none of the physiologic and metabolic fluctuations exhibited in controls. We conclude that neither the tested vectors nor great excesses of circulating BChE affect general physiology directly, while they protect mice from disturbance by cocaine. Hence, viral gene transfer of BChE appears benign and worth exploring as a therapy for cocaine abuse and possibly other disorders as well.

Combinatorial treatment with oncolytic adenovirus and helper-dependent adenovirus augments adenoviral cancer gene therapy

Oncolytic adenoviruses (Onc.Ads) produce significant antitumor effects but as single agents they rarely eliminate tumors. Investigators have therefore incorporated sequences into these vectors that encode immunomodulatory molecules to enhance antitumor immunity. Successful implementation of this strategy requires multiple tumor immune inhibitory mechanisms to be overcome, and insertion of the corresponding multiple functional genes reduces the titer and replication of Onc.Ads, compromising their direct ant-tumor effects. By contrast, helper-dependent (HD) Ads are devoid of viral coding sequences, allowing inclusion of multiple transgenes. HDAds, however, lack replicative capacity. Since HDAds encode the adenoviral packaging signal, we hypothesized that the coadministration of Onc.Ad with HDAd would allow to be amplified and packaged during replication of Onc.Ad in transduced cancer cells. This combination could provide immunostimulation without losing oncolytic activity. We now show that coinfection of Onc.Ad with HDAd subsequently replicates HDAd vector DNA in trans in human cancer cell lines in vitro and in vivo, amplifying the transgenes the HDAd encode. This combinatorial treatment significantly suppresses the tumor growth compared to treatment with a single agent in an immunocompetent mouse model. Hence, combinatorial treatment of Onc.Ad with HDAd should overcome the inherent limitations of each agent and provide a highly immunogenic oncolytic therapy.

Gene Transfer of Mutant Mouse Cholinesterase Provides High Lifetime Expression and Reduced Cocaine Responses with No Evident Toxicity

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with 3H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3×1011 particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (1010 particles) to 20,000 fold (1013 particles), while the hdAD vector (1.7×1012 particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7×1012 particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose.

Vector and helper genome rearrangements occur during production of helper-dependent adenoviral vectors.

Helper-dependent adenoviral vectors (HD Ad) hold extreme promise for gene therapy of human diseases. All viral genes are deleted in HD Ad vectors, and therefore, the presence of a helper virus is required for their production. Current methods to minimize helper contamination in large-scale preparations rely on the use of the Cre/loxP system. The inclusion of loxP sites flanking the packaging signal results in its excision in the presence of Cre recombinase, preventing helper genome encapsidation. It is well established that the level of Cre recombinase activity is important in determining the degree of helper contamination. However, there is little information on other mechanisms that could also play an important role. We have generated several HD Ad vectors containing a rapalog-inducible system to regulate transgene expression, or LacZ under the control of the elongation factor 1 α promoter. Large-scale production of these vectors resulted in abundant helper contamination. Viral DNA analysis revealed the presence of rearrangements between vector and helper genomes. The rearrangements involved a helper DNA molecule with a fragment of the left arm of the HD Ad vector, including its ITR, packaging signal, and some stuffer sequence. Overall, our data suggest that helper DNA molecules that accumulate after Cre recombinase activity are prone to rearrangements, resulting in helper genomes that have incorporated a packaging signal from the vector. Helper particles with rearranged genomes have a growth advantage. This study identifies a novel mechanism leading to helper contamination during helper-dependent adenoviral vector production.

Rescue administration of a helper-dependent adenovirus vector with long-term efficacy in dogs with glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia.

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2- to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.

IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis

Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69-CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.

A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.

676. AAV-Rep 78 Mediated Rescue and Integration of a 27kb Transgene Cassette

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. AAV Rep78 is able to target AAV integration vectors to a specific site on human chromosome 19 called AAVS1. We studied whether this ability can be harnessed to achieve site-specific integration of a 27 kb transgene cassette into a model cell line for hematopoietic cells (Mo7e). To deliver Rep78 and the transgene to MO7e cells we used helper-dependent adenovirus vectors containing Ad serotype 35 fibers (HD-Ad). An HD-Ad containing the rep78 gene conferred efficient Rep 78 expression in target cells. Upon co-infection of HD-Ad-rep78 with an HD-Ad vector containing a 27k |[beta]|-globin-LCR-GFP transgene cassette flanked by AAV ITRs (HD-Ad.AAV-LCR), efficient Rep78-mediated rescue/excision of the transgene cassette was observed in 293 cells but not in MO7e cells. We found that rescue in 293 cells was enhanced due to expression of viral proteins, specifically proteins encoded by Ad5 E2a and E4orf6. Although no rescue of the LCR-GFP cassette was detectable in MO7e cells, co-infection of HD-Ad.rep78 increased the overall stable transduction frequency of HD.Ad.AAV-LCR and mediated integration of the transgene cassette into AAV-S1. (40% of all analyzed vector junction were in AAVS1). Interestingly, analysis of integration junctions revealed that Ad integration occurred in 80% of sites via the Ad ITR and only in 20% of sites via the AAV ITR. No rearrangements within AAV-S1 or AAV-S1 specific transgene integration were observed when HD- Ad.AAV-LCR was infected onto MO7e cells without the Rep78-expressing HD Ad vector. Our data indicate that rescue of the AAV-ITR flanked transgene cassette is not necessary for integration into AAV-S1. We are currently evaluating whether rescue can enhance the frequency of site-specific integration. This study provides a basis for the construction of vectors that allow for preferential site-specific transgene integration in hematopoietic cells.

211. Development of Helper-Dependent Adenovirus (HDAd) Vectors for Gene Therapy in the Mouse Model for Glycogen Storage Disease Type Ia (GSD-Ia)

Objective: The deficiency of glucose-6-phosphatase (G6Pase) underlies glycogen storage disease type Ia (GSD-Ia, also known as von Gierke disease; MIM 232200), an autosomal recessive disorder of metabolism associated with life-threatening hypoglycemia and growth retardation. Regulation of G6Pase at the level of transcription occurs in response to glucose and insulin levels. We investigated the hypothesis that G6Pase expression with an HDAd vector containing a glucose-response promoter would correct hypoglycemia and prolong survival in GSD-I mice.

46. Clinical and Functional Recovery from Experimental Autoimmune Encephalomyelitis by Intracisternal Delivery of IL4 from a Helper-Dependent Adenoviral Vector. A Pre-Clinical Model of Gene Therapy of Multiple Sclerosis

Widespread demyelination, axonal loss and permanent neurological impairment are hallmarks of chronic, inflammatory demyelinating disorders of the CNS, such as multiple sclerosis (MS). The multifocal, recurrent nature of MS complicates cell and gene therapy approaches aimed at restoring axonal conduction by inducing re-myelination, or transplanting myelin-forming cells, at the sites of injury. Previous studies have demonstrated that systemic delivery of the anti-inflammatory cytokine IL4 to the CNS through the cerebro-spinal fluid circulation can prevent demyelination in experimentally-induced autoimmune encephalomyelitis (EAE), a murine model of MS (Hum Gene Ther 8:13, 2001). We have established a cytokine-delivery system to the CNS by transducing the ependimal layer lining the ventricular system with fully deleted, helper-dependent adenoviral vectors (HD-Ad). A single injection of a HD-Ad vector expressing IL-4 and GFP (AdIL-4G) into the cisterna magna of immunocompetent mice is sufficient to transduce ependimal and leptomeningeal cells surrounding ventricular and sub-arachnoid spaces. Vector DNA and GFP expression were detected in ependimal and leptomeningeal cells by real-time PCR after laser-capture microdissection and immunohystochemistry up to five months after injection. IL-4 levels up to 340 pg/ml were measured by ELISA in the cerebrospinal fluid five months after treatment. Serum IL-4 levels were always below detection limits. To assess the therapeutic potential of intracysternal IL-4 delivery, 30 mice affected by myelin oligodendrocyte glycoprotein (MOG35-55)-induced EAE were injected the day of clinical onset with escalating doses (105 to 107 i.p./mouse) of AdIL-4G or a control HD-Ad vector expressing only GFP. Mice treated with >106 i.p. of AdIL-4G displayed a progressive clinical recovery, and 80 days after immunization had an average EAE score of 1.0 (±1.0) vs. 1.62 (±0.4) in control mice (p<0.05). Motor evoked potentials (MEP) were recordable in 50% of the control mice, and in all but one of the IL-4-treated mice (p<0.05). In mice were MEP were recordable, central conduction time was significant decreased in IL-4 treated vs. control mice (p<0.05). IL-4 gene therapy by intracisternal administration of HD-Ad vectors therefore induces clinical and functional recovery in a murine model of MS. Biodistribution, immunological and toxicity studies in non-human primates are currently in progress.

Primary and secondary prevention in hyperlipidaemic patients. Their compliance with directions and treatment given, according to the records of our clinics of hyperlipidaemias

In continuing efforts to develop gene transfer of human butyrylcholinesterase (BChE) as therapy for cocaine addiction, we conducted wide-ranging studies of physiological and metabolic safety. For that purpose, mice were given injections of adeno-associated virus (AAV) vector or helper-dependent adenoviral (hdAD) vector encoding human or mouse BChE mutated for optimal cocaine hydrolysis. Age-matched controls received saline or AAV-luciferase control vector. At times when transduced BChE was abundant, physiologic and metabolic parameters in conscious animals were evaluated by non-invasive Echo-MRI and an automated “Comprehensive Laboratory Animal Monitoring System” (CLAMS). Despite high vector doses (up to 1013 particles per mouse) and high levels of transgene protein in the plasma (∼1500-fold above baseline), the CLAMS apparatus revealed no adverse physiologic or metabolic effects. Likewise, body composition determined by Echo-MRI, and glucose tolerance remained normal. A CLAMS study of vector-treated mice given 40 mg/kg cocaine showed none of the physiologic and metabolic fluctuations exhibited in controls. We conclude that neither the tested vectors nor great excesses of circulating BChE affect general physiology directly, while they protect mice from disturbance by cocaine. Hence, viral gene transfer of BChE appears benign and worth exploring as a therapy for cocaine abuse and possibly other disorders as well.

Production of helper-dependent adenovirus vector relies on helper virus structure and complementing.

The helper-dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD-Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD-Ad vector is generally lower than that of an E1-deleted first-generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD-Ad vector. To study this question and improve HD-Ad vector production, we have generated four different helper viruses with a wild-type or deleted E3 region, and with a relocated loxP. We have also constructed a first-generation vector with a wild-type E3 region and without the loxP site. We compared the replication of these viruses in Cre-positive and -negative cells and studied their complementing for HD-Ad vector production. Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD-Ad vector was obtained when a helper virus with wild-type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability. Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD-Ad vector can be further improved by modifications in helper virus structure.

Generation of helper-dependent adenoviral vectors by homologous recombination.

Helper-dependent adenoviral vectors (HD-Ad) represent a potentially valuable tool for safe and prolonged gene expression in vivo. The current approach for generating these vectors is based on ligation of the expression cassette into large plasmids containing the viral inverted terminal repeats flanking "stuffer" DNA to maintain a final size above the lower limit for efficient packaging into the adenovirus capsid (approximately 28 kb). The ligation to produce the viral plasmid is generally very inefficient. Similar problems in producing first-generation adenoviral (FG-Ad) vectors were circumvented with the development of a system taking advantage of efficient homologous recombination between a shuttle plasmid containing the expression cassette and a FG-Ad vector backbone in the Escherichia coli strain BJ5183. Here we describe a method for fast and efficient generation of HD-Ad vector plasmids that can accommodate expression cassettes of any size up to 35 kb. To validate the system, we generated a HD-Ad vector expressing the fusion protein between beta-galactosidase and neomycin resistance genes under the control of the SR alpha promoter, and one expressing the enhanced green fluorescent protein under the control of the cytomegalovirus promoter. The viruses were rescued and tested in vitro and for in vivo expression in mice. The data collected indicate the possibility for achieving a high level of hepatocyte transduction using HD-Ad vectors derived from plasmids obtained by homologous recombination in E. coli, with no significant alteration of liver enzymes and a less severe, transient thrombocytopenia in comparison with previous reports with similar doses of a FG-Ad vector.

Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector.

Ideally, somatic gene therapy should result in lifetime reversal of genetic deficiencies. However, to date, phenotypic correction of monogenic hyperlipidemia in mouse models by in vivo gene therapy has been short-lived and associated with substantial toxicity. We have developed a helper-dependent adenoviral vector (HD-Ad) containing the apolipoprotein (apo) E gene. A single i.v. injection of this vector completely and stably corrected the hypercholesterolemia in apoE-deficient mice, an effect that lasted the natural lifespan of the mice. At 2.5 years, control aorta was covered 100% by atherosclerotic lesion, whereas aorta of treated mice was essentially lesion-free. There was negligible toxicity associated with the treatment. We also developed a method for repeated HD-Ad vector administration that could be applied to organisms, e.g., humans, with life spans longer than 2-3 years. These studies indicate that HD-Ad is a promising system for liver-directed gene therapy of metabolic diseases.

Prolonged transgene expression mediated by a helper-dependent adenoviral vector (hdAd) in the central nervous system.

Conventional adenoviral vectors such as E1-deleted first-generation adenovirus (fgAd) elicit striking host immune response, resulting in limited expression of the transgene. A recently described helper-dependent, or gutless, adenoviral vector (hdAd) can promote stable transgene expression in peripheral organs, including the liver. We therefore investigated the safety and durability of hdAd-mediated gene transfer to the central nervous system (CNS) of rats compared with gene delivery by fgAd. Equal amounts of either fgAd or hdAd carrying the beta geo transgene were stereotactically injected into the right hippocampus of adult rats. Transgene expression was assessed by histochemical staining, transgene stability by PCR analysis, and immune infiltration of T lymphocytes and macrophages by immunocytochemical methods. Strong transgene expression from either vector was detected in brain tissue examined on day 6 postinoculation. Thereafter, fgAd-mediated gene expression rapidly decreased, becoming undetectable by day 66, while expression from the hdAd vector persisted throughout the test period. PCR confirmed the presence of hdAd-associated DNA at 66 days postinoculation. The hdAd injection elicited apparently lower numbers of brain-infiltrating macrophages and T cells than did administration of fgAd. These results indicate improved transgene expression and reduced immunogenicity with use of hdAd to deliver genes to the CNS.