Showing papers in "Human Gene Therapy in 2001"

Nonviral Vectors in the New Millennium: Delivery Barriers in Gene Transfer

Abstract: Development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired diseases by gene therapy. To this end, various nonviral vectors have been designed and developed, and some of them are in clinical trials. The simplest approach is naked DNA injection into local tissues or systemic circulation. Physical (gene gun, electroporation) and chemical (cationic lipid or polymer) approaches have also been utilized to improve the efficiency and target cell specificity of gene transfer by plasmid DNA. After administration, however, nonviral vectors encounter many hurdles that result in diminished gene transfer in target cells. Cationic vectors sometimes attract serum proteins and blood cells when entering into blood circulation, which results in dynamic changes in their physicochemical properties. To reach target cells, nonviral vectors should pass through the capillaries, avoid recognition by mononuclear phagocytes, emerge from the blood vessels to the interstitium, and bind to the surface of the target cells. They then need to be internalized, escape from endosomes, and then find a way to the nucleus, avoiding cytoplasmic degradation. Successful clinical applications of nonviral vectors will rely on a better understanding of barriers in gene transfer and development of vectors that can overcome these barriers.

Systematic Determination of the Packaging Limit of Lentiviral Vectors

Abstract: Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.

Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column.

Abstract: One of the most promising gene transfer vectors in human clinical trials is AAV2. The quality of the vector preparations is a key element in obtaining reliable and reproducible data in preclinical studies. However, established protocols either result in impure, low infectious virus (CsCl2 gradient centrifugation) or demand a high level of manual and technical skills (CsCl2 gradient centrifugation, iodixanol/heparin or HPLC purification). In this study, we present an easy-to-do single-step column purification (SSCP) of AAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation. Various vector preparations generated by our method reproducibly showed high titers, infectivity, and purity. In vivo, our single-step column-purified AAV2 vectors mediate significantly higher transduction efficiency compared with conventional protocols. Investigators still unsatisfied with previously published techniques or new to the field of AAV production may find in our method an interesting alternative.

A Phase I Study of Aerosolized Administration of tgAAVCF to Cystic Fibrosis Subjects with Mild Lung Disease

Abstract: Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.

Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation.

Abstract: This gene transfer experiment is the first Parkinson's Disease (PD) protocol to be submitted to the Recombinant DNA Advisory Committee. The principal investigators have uniquely focused their careers on both pre-clinical work on gene transfer in the brain and clinical expertise in management and surgical treatment of patients with PD. They have extensively used rodent models of PD for proof-of-principle experiments on the utility of different vector systems. PD is an excellent target for gene therapy, because it is a complex acquired disease of unknown etiology (apart from some rare familial cases) yet it is characterized by a specific neuroanatomical pathology, the degeneration of dopamine neurons of the substantia nigra (SN) with loss of dopamine input to the striatum. This pathology results in focal changes in the function of several deep brain nuclei, which have been well-characterized in humans and animal models and which account for many of the motor symptoms of PD. Our original approaches, largely to validate in vivo gene transfer in the brain, were designed to facilitate dopamine transmission in the striatum using an AAV vector expressing dopamine-synthetic enzymes. Although these confirmed the safety and potential efficacy of AAV, complex patient responses to dopamine augmenting medication as well as poor results and complications of human transplant studies suggested that this would be a difficult and potentially dangerous clinical strategy using current approaches. Subsequently, we and others investigated the use of growth factors, including GDNF. These showed some encouraging effects on dopamine neuron survival and regeneration in both rodent and primate models; however, uncertain consequences of long-term growth factor expression and question regarding timing of therapy in the disease course must be resolved before any clinical study can be contemplated. We now propose to infuse into the subthalamic nucleus (STN) recombinant AAV vectors expressing the two isoforms of the enzyme glutamic acid decarboxylase (GAD-65 and GAD-67), which synthesizes the major inhibitory neurotransmitter in the brain, GABA. The STN is a very small nucleus (140 cubic mm or 0.02% of the total brain volume, consisting of approximately 300,000 neurons) which is disinhibited in PD, leading to pathological excitation of its targets, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr). Increased GPi/SNpr outflow is believed responsible for many of the cardinal symptoms of PD, i.e., tremor, rigidity, bradykinesia, and gait disturbance. A large amount of data based on lesioning, electrical stimulation, and local drug infusion studies with GABA-agonists in human PD patients have reinforced this circuit model of PD and the central role of the STN. Moreover, the closest conventional surgical intervention to our proposal, deep brain stimulation (DBS) of the STN, has shown remarkable efficacy in even late stage PD, unlike the early failures associated with recombinant GDNF infusion or cell transplantation approaches in PD. We believe that our gene transfer strategy will not only palliate symptoms by inhibiting STN activity, as with DBS, but we also have evidence that the vector converts excitatory STN projections to inhibitory projections. This additional dampening of outflow GPi/SNpr outflow may provide an additional advantage over DBS. Moreover, of perhaps the greatest interest, our preclinical data suggests that this strategy may also be neuroprotective, so this therapy may slow the degeneration of dopaminergic neurons. We will use both GAD isoforms since both are typically expressed in inhibitory neurons in the brain, and our data suggest that the combination of both isoforms is likely to be most beneficial. Our preclinical data includes three model systems: (1) old, chronically lesioned parkinsonian rats in which intraSTN GAD gene transfer results not only in improvement in both drug-induced asymmetrical behavior (apomorphine symmetrical rotations), but also in spontaneous behaviors. In our second model, GAD gene transfer precedes the generation of a dopamine lesion. Here GAD gene transfer showed remarkable neuroprotection. Finally, we carried out a study where GAD-65 and GAD-67 were used separately in monkeys that were resistant to MPTP lesioning and hence showed minimal symptomatology. Nevertheless GAD gene transfer showed no adverse effects and small improvements in both Parkinson rating scales and activity measures were obtained. In the proposed clinical trial, all patients will have met criteria for and will have given consent for STN DBS elective surgery. Twenty patients will all receive DBS electrodes, but in addition they will be randomized into two groups, to receive either a solution containing rAAV-GAD, or a solution which consists just of the vector vehicle, physiological saline. Patients, care providers, and physicians will be blind as to which solution any one patient receives. All patients, regardless of group, will agree to not have the DBS activated until the completion and unblinding of the study. Patients will be assessed with a core clinical assessment program modeled on the CAPSIT, and in addition will also undergo a preop and several postop PET scans. At the conclusion of the study, if any patient with sufficient symptomatic improvement will be offered DBS removal if they so desire. Any patients with no benefit will simply have their stimulators activated, which would normally be appropriate therapy for them and which requires no additional operations. If any unforeseen symptoms occur from STN production of GABA, this might be controlled by blocking STN GABA release with DBS, or STN lesioning could be performed using the DBS electrode. Again, this treatment would not subject the patient to additional invasive brain surgery. The trial described here reflects an evolution in our thinking about the best strategy to make a positive impact in Parkinson Disease by minimizing risk and maximizing potential benefit. To our knowledge, this proposal represents the first truly blinded, completely controlled gene or cell therapy study in the brain, which still provides the patient with the same surgical procedure which they would normally receive and should not subject the patient to additional surgical procedures regardless of the success or failure of the study. This study first and foremost aims to maximally serve the safety interests of the individual patient while simultaneously serving the public interest in rigorously determining in a scientific fashion if gene therapy can be effective to any degree in treating Parkinson's disease.

Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates.

Abstract: We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 ± 187 ng/g for the arm and 1692 ± 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of β-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in huma...

CMV-β-Actin Promoter Directs Higher Expression from an Adeno-Associated Viral Vector in the Liver than the Cytomegalovirus or Elongation Factor 1α Promoter and Results in Therapeutic Levels of Human Factor X in Mice

Abstract: Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken β-actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1α promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-β-actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice....

A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis.

Abstract: Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-α, or IFN-γ became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken f...

Insertional Mutagenesis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors Targeted to Alternative Cell-Surface Receptors

Abstract: Recombinant adeno-associated virus (AAV) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. However, AAV-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (HSPG), the primary cellular receptor for the virus. Conversely, an increasing number of cell types are being identified that do not express HSPG and are therefore poor targets for AAV-mediated gene transfer. To address these issues, we have developed strategies to physically modify AAV vectors and allow efficient, HSPG-independent, receptor-targeted infection. We began by generating a series of 38 virus capsid mutants containing peptide insertions at 25 unique sites within the AAV capsid protein. The mutant viruses were characterized on the basis of their phenotypes and grouped into three classes: class I mutants (4 of 38) did not assemble particles; class II mutants (14 of 38) assembled noninfectious particles; and class III mutants (20 of 38) assembled fully infectious particles. We examined the HSPG-binding characteristics of the class II mutants and showed that a defect in receptor binding was a common reason for their lack of infectivity. The display of foreign peptide epitopes on the surface of the mutant AAV particles was found to be highly dependent on the inclusion of appropriate scaffolding sequences. Optimal scaffolding sequences and five preferred sites for the insertion of targeting peptide epitopes were identified. These sites are located within each of the three AAV capsid proteins, and thus display inserted epitopes 3, 6, or 60 times per vector particle. Modified AAV vectors displaying a 15-amino acid peptide, which binds to the human luteinizing hormone receptor (LH-R), were generated and assessed for their ability to target gene delivery to receptor-bearing cell lines. Titers of these mutant vectors were essentially the same as wild-type vector. The LH-R-targeted vector was able to transduce ovarian cancer cells (OVCAR-3) in an HSPG-independent manner. Furthermore, transduction was shown to proceed via the LH-R and therefore treatment of OVCAR-3 cells with progesterone, to increase LH-R expression, accordingly increased LH mutant-mediated gene transfer. This technology may have a significant impact on the use of AAV vectors for human gene therapy.

Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression

Abstract: Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

Abstract: By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Baboon Mesenchymal Stem Cells Can Be Genetically Modified to Secrete Human Erythropoietin In Vivo

Abstract: Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

A New-Generation Stable Inducible Packaging Cell Line for Lentiviral Vectors

Abstract: We have successfully generated and characterized a stable packaging cell line for HIV-1-based vectors. To allow safe production of vector, a minimal packaging construct carrying only the coding sequences of the HIV1 gag‐pol, tat, and rev genes was stably introduced into 293G cells under the control of a Tet o minimal promoter. 293G cells express the chimeric Tet R /VP16 trans-activator and contain a tetracycline-regulated vesicular stomatitis virus protein G (VSV-G) envelope gene. When the cells were grown in the presence of tetracycline the expression of both HIV-1-derived and VSV-derived packaging functions was suppressed. On induction, approximately 50 ng/ml/24 hr of Gag p24 equivalent of vector was obtained. After introduction of the transfer vector by serial infection, vector could be collected for several days with a transduction efficiency similar or superior to that of vector produced by transient transfection both for dividing and growth-arrested cells. The vector could be effectively concentrated to titers reaching 10 9 transducing units/ml and allowed for efficient delivery and stable expression of a GFP transgene in the mouse brain. The packaging cell line and all vector producer clones described here were shown to be free from replication-competent recombinants, and from recombinants between packaging and vector constructs that transfer the viral gag‐pol genes. The packaging cell line and the assays developed will advance lentiviral vectors toward the stringent requirements of clinical applications.

A High-Efficiency Synthetic Promoter That Drives Transgene Expression Selectively in Noradrenergic Neurons

Abstract: Gene promoter systems that drive high-level, long-term, and cell-specific transgene expression are of great interest because of their potential utility for gene therapy. To generate an efficient promoter system specific for noradrenergic (NA) neurons, we multimerized an NA-specific cis-regulatory element (PRS2) identified in the human dopamine beta-hydroxylase (hDBH) promoter, and combined it with a minimal promoter (containing a TATA box and transcription start site). Forms of this synthetic promoter that contain 8 or more copies of PRS2 were >50 times more effective than the 1.15-kb hDBH promoter at driving reporter gene expression in cell lines originated from NA neurons. Neither the synthetic promoter nor the 1.15-kb hDBH promoter drove reporter gene expression in nonneuronal cells. Microinjections of an adenoviral vector containing the synthetic promoter directly into rat brain caused more strict NA-specific reporter gene expression than that caused by a vector containing the 1.15-kb hDBH promoter when the targeted region contained large numbers of NA neurons (locus coeruleus). Furthermore, the vector containing the synthetic promoter caused less nonspecific ("leaky") reporter gene expression than that caused by the vector containing the 1.15-kb hDBH promoter when the targeted region was devoid of NA neurons (cerebellum, dentate gyrus). Together, these studies provide in vitro and in vivo evidence that this novel synthetic promoter can target transgene expression to NA neurons even more efficiently and selectively than the naturally occurring, 1.15-kb hDBH promoter.

Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells.

Abstract: Myoblast transplantation has been investigated as a therapy for muscle-related diseases and as a gene delivery vehicle for therapeutic recombinant proteins. Clinical successes involving muscle cell transplantation have been limited, in part because of poor donor cell survival, and the heterogeneous nature of myogenic donor cells has largely been ignored. We have previously reported an isolation technique, preplating, that results in purified myogenic cells that are capable of significantly higher rates of donor cell survival leading to enhanced gene transfer to skeletal muscle. Characterization of these purified cells revealed that they display markers common to stem cells and are capable of multilineage differentiation. This study was performed to phenotypically characterize, by flow cytometry, muscle-derived cell populations obtained by the preplate technique for the purpose of eventually developing a method to quickly identify and isolate viable muscle cells best suited for transplantation. Muscle cell cultures were analyzed for expression of the surface proteins Sca-1, c-Kit, and CD34. We found that the preplate technique purifies distinct myogenic cell subpopulations expressing CD34 alone (Sca-1 negative) and Sca-1 alone (CD34 negative), but that this expression is subject to change with time in culture. Isolation and transplantation of phenotypically pure Sca-1-positive myogenic cells, obtained by magnetic cell sorting, demonstrates the ability to quickly select viable myogenic cells capable of regenerating skeletal muscle and restoring dystrophin expression within dystrophic host skeletal muscle. Flow cytometric described phenotypes will aid in the rapid isolation of specific donor cell populations for muscle cell transplants and muscle cell-mediated gene therapies, thereby enhancing their future success.

Clinical protocol. An open-label, phase I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD).

Abstract: Age-Related Macular Degeneration (AMD) is, together with Diabetic Retinopathy, the most common cause of vision loss among adults in the U.S. and other developed countries. In the U.S., at least 1.7 million people have impaired vision due to AMD. Every year, more than 165,000 people contract AMD and 16,000 go blind from it, predominantly from a rapidly progressing form of the disease called "wet" AMD. Wet AMD is characterized by serious or hemorrhagic detachment of the retinal pigment epithelium and choroidal neovascularization. The macula has the highest concentration of photoreceptors facilitating central vision and permitting high-resolution visual acuity. The damage caused by the leakage and fibrovascular scarring in wet AMD leads to profound loss of central vision and severe loss of visual acuity (usually 20/200 or worse). People with wet AMD have several limitations, including inability to read, inability to recognize faces or drive, and the disease often leads to blindness. The onset of severe visual changes in wet AMD can occur suddenly. More than 400,000 Americans are currently affected by this form of the disease, and the incidence is rising rapidly with the aging of the population. Therefore, the serious consequences of this disease along with the limited treatment options and their effectiveness make this a very good candidate for a gene transfer treatment approach. The investigational agent, Ad(GV)PEDF.11D, is an E1-, partial E3-, E4- deleted replication-deficient, adenovirus serotype 5, gene transfer vector. The transgene in this vector is the cDNA for human pigment epithelium-derived factor (PEDF). PEDF is one of the most potent known antiangiogenic proteins found in humans. While Ad(GV)PEDF.11D is able to transduce many somatic cell types, the natural barrier to other tissues created by the retina limits the ability of Ad(GV)PEDF.11D to affect tissues other than in the eye. Intravitreal administration of Ad(GV)PEDF.11D provides a convenient means of delivering PEDF to the relevant cells within the eye likely to result in a more prolonged duration of effect versus administration of the PEDF protein alone. In three murine disease models (the laser-induced choroidal neovascularization model, the VEGF transgenic model, and the retinopathy of prematurity model) significant inhibition of neovascularization (up to 85%) was demonstrated with doses of Ad(GV)PEDF vectors ranging from 1 x 10(8) to 1 x 10(9) pu. In toxicology studies performed in Cynomolgus monkeys, a dose-related inflammatory response was observed. A dose of 1 x 10(8) pu caused no adverse effects, while the inflammatory response observed at 1 x 10(9) pu was minimal and fully reversible. The observed inflammatory response at doses of 1 x 10(10) and 5 x 10(10) pu were increasingly severe. The proposed clinical trial is an open-label, dose-escalation, phase I study to investigate the safety, tolerability and potential activity of intravitreal injection of Ad(GV)PEDF.11D in patients with wet AMD. Ad(GV)PEDF.11D will be injected once via intravitreal injection into the eye with the most advanced AMD based on visual acuity. Subjects will be age 50 or over and have severe wet AMD in at least one eye defined by a best-corrected vision of 20/200 or worse. The primary objectives of this investigation are: (1) to assess the safety, tolerability and feasibility of intravitreal injection of Ad(GV)PEDF.11D in patients with severe, neovascular AMD, (2) to identify the maximum tolerated dose (MTD) of Ad(GV)PEDF.11D, and (3) to get some indication of potential activity of Ad(GV)PEDF.11D.

Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study.

Abstract: A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by ...

In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors

Abstract: Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro. However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system. To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle. Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Two viruses were constructed to carry either the Escherichia coli β-galactosidase (β-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer. The greater gene transduction efficiency of the Bac-G-βGal vector was confirmed by comparing the β-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-βGal, a vector that lacks VSV-G. Similarly, a 5- to 10-fold increa...

Muscle-Specific Promoters May Be Necessary for Adeno-Associated Virus-Mediated Gene Transfer in the Treatment of Muscular Dystrophies

Abstract: Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg-/- mice) γ-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg-/- mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of γ-sarcoglycan in myofibers after direct muscle injection into gsg-/- mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the γ-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when γ-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to γ-sarcoglycan. When using an rAAV vector, expressing the high...

Transfer of heme oxygenase 1 cDNA by a replication-deficient adenovirus enhances interleukin 10 production from alveolar macrophages that attenuates lipopolysaccharide-induced acute lung injury in mice.

Abstract: By using a direct, intratracheal inoculation of an adenovirus encoding heme oxygenase 1 (Ad.HO-1), model gene therapy for acute lung injury induced by inhaled pathogen was performed. Data demonstrated that Ad.HO-1 administration is as effective as the pharmacologic upregulation of the endogenous HO-1 gene expression by hemin to attenuate neutrophilic inflammations of the lung after aerosolized lipopolysaccharide (LPS) exposure. Interestingly, immunohistochemical analysis revealed that the HO-1 gene was transferred not only to the airway epithelium, but to the alveolar macrophages (AMs). Moreover, overexpression of exogenous HO-1 in the macrophages provided a high level of endogenous interleukin 10 (IL-10) production from the macrophages, and additional experiments using IL-10 knockout mice demonstrated that the increase in IL-10 in the macrophages was critical for the resolution of neutrophilic migration in the lung after LPS exposure. These results suggest that AMs not only are barriers for efficient gene transfer to the respiratory epithelium, but also represent logical targets for Ad-mediated, direct, in vivo gene therapy strategies for inflammatory disorders in humans.

Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma.

Abstract: Replication-competent, attenuated herpes simplex viruses (HSV) have been demonstrated to be effective oncolytic agents in a variety of malignant tumors. Cytokine gene transfer has also been used as immunomodulatory therapy for cancer. To test the utility of combining these two approaches, two oncolytic HSV vectors (NV1034 and NV1042) were designed to express the murine GM-CSF and murine IL-12 genes, respectively. These cytokine-carrying variants were compared with the analogous non-cytokine-carrying control virus (NV1023) in the treatment of murine SCC VII squamous cell carcinoma. All three viruses demonstrated similar infection efficiency, viral replication, and cytotoxicity in vitro. SCC VII cells infected by NV1034 and NV1042 effectively produced GM-CSF and IL-12, respectively. In an SCC VII subcutaneous flank tumor model in immunocompetent C3H/HeJ mice, intratumoral injection with each virus caused a significant reduction in tumor volume compared with saline injections. The NV1042-treated tumors showe...

Cochlear Gene Delivery through an Intact Round Window Membrane in Mouse

Abstract: Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors.

Abstract: Clinical studies were performed with a recombinant mutant adenovirus with an E1B 55-kDa deletion, dl1520, to assess its toxicity and efficacy in patients with irresectable primary and secondary liver tumors. A phase I study showed that dl1520 was well tolerated when administered directly intratumorally, intraarterially, or intravenously up to a dose of 3 x 10(11) PFU. Ultrastructural examination of tissue showed the presence of adenovirus in cell cytoplasm around the nucleus and revealed two dissimilar end points of cell death after virus infection: a preapoptotic sequence and necrosis. A phase II study showed that the combination of dl1520 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well tolerated. Further improvement in the recombinant vector design will be needed in order to achieve better clinical response.

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization.

Abstract: Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothe...

Use of viral fusogenic membrane glycoproteins as novel therapeutic transgenes in gliomas.

Abstract: Malignant gliomas are the most common primary brain tumors in adults and, with few exceptions, have a dismal prognosis despite the therapeutic use of surgery, radiation therapy, and chemotherapy. Because CNS gliomas rarely metastasize, they represent an attractive target for gene therapy through local gene delivery. Here we report on the use of two different fusogenic membrane glycoproteins (FMGs), the measles virus proteins F and H (MV-F and MV-H) and a mutated form of the retroviral envelope protein of the gibbon ape leukemia virus (GALV.fus), as a novel class of therapeutic transgenes in gliomas. Transfection of U87 and U118 cells with MV-F and MV-H cDNA or GALV.fus cDNA led in 48 hr to massive syncytial formation followed by cell death. FMG-mediated cytotoxicity in the U87 and U118 cell lines was superior to the cytotoxicity caused by transfection with HSV-tk cDNA followed by ganciclovir (GCV) treatment at all time points. At high-density cell seeding, addition of tumor cells transfected with MV-F and H killed at least 1 log more cells than by HSV-tk + GCV treatment, indicating higher bystander effect. Similar results were obtained with GALV.fus. The mechanism of syncytial death in cultured glioma cell lines was predominantly apoptotic. Transfection of U87 cells with F + H or GALV.fus expression constructs completely suppressed their tumorigenicity. Treatment of established U87 xenografts in nude mice with a combination of F and H adenoviruses at 1:1 ratio led to complete tumor regression, significantly higher antitumor effect, and prolongation of survival as compared with control animals treated with a GFP adenovirus. In summary, the viral fusogenic membrane glycoproteins (GALV and the MV-F + MV-H combination) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas.

Matrix Immobilization Enhances the Tissue Repair Activity of Growth Factor Gene Therapy Vectors

Abstract: Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p # 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold ( p , 0.009), respectively, and wound closure up to 2-fold ( p , 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGFBB protein were required for neotissue induction approaching equivalence to a single application of collagenimmobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus

Abstract: Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice.

Abstract: TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.

Preexisting Antiadenoviral Immunity Is Not a Barrier to Efficient and Stable Transduction of the Brain, Mediated by Novel High-Capacity Adenovirus Vectors

Abstract: The utility of first-generation adenovirus vectors for long-term gene transfer in humans is limited by preexisting antiadenoviral immunity. We demonstrate here that new-generation high-capacity adenovirus vectors (HC-Ads) can efficiently transduce the brain and mediate stable transgene expression for at least 2 months, even in the presence of a preexisting antiadenoviral immune response. First-generation vector-mediated transduction was almost completely abolished in preimmunized animals within 60 days of the vector injection. Levels of HC-Ad-mediated transduction by 3 days postinjection were not significantly affected by preimmunization, were reduced within 14 days to 56% of those levels seen in nonimmunized animals, and remained stable until day 60 postinjection. Acute brain inflammation elicited by the HC-Ad vector injection was more transient, and was reduced in intensity compared with brain inflammation elicited by the first-generation vector injection in immunized animals. Inflammation was significantly higher in all immunized animals than in nonimmunized animals. Our results show that preexisting antiadenoviral immunity does not significantly reduce initial HC-Ad-mediated infection of the brain and is not a barrier to stable HC-Ad vector-mediated transduction of the CNS. Although input HC-Ad capsid proteins injected into the brain may contain transient targets for a brain-infiltrating cellular adenovirus-specific immune response, this fails to eliminate transgene expression. Thus HC-Ads show promise for gene therapy of chronic brain disease.

Wild-type adenovirus decreases tumor xenograft growth, but despite viral persistence complete tumor responses are rarely achieved--deletion of the viral E1b-19-kD gene increases the viral oncolytic effect.

Abstract: Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was ...