Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa.

Mutations of the LAMB3 gene cause a lethal form of junctional epidermolysis bullosa (JEB). We hypothesized that early intra-amniotic gene transfer in a severe murine model of JEB would improve or correct the skin phenotype. Time-dated fetuses from heterozygous LAMB3(IAP) breeding pairs underwent ultrasound guided intra-amniotic injection of lentiviral vector encoding the murine LAMB3 gene at embryonic day 8 (E8). Gene expression was monitored by immunohistochemistry. The transgenic laminin-ß3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-332 in the basement membrane zone of skin and mucosa. Ultrastructually, the restoration of ∼60% of hemidesmosomal structures was also noted. Although we could correct the skin phenotype in 11.9% of homozygous LAMB3(IAP) mice, none survived beyond 48 h. However, skin transplants from treated E18 homozygous LAMB3(IAP) fetuses maintained normal appearance for 6 months with persistence of normal assembly of laminin-332. These results demonstrate for the first time long-term phenotypic correction of the skin pathology in a severe model of JEB by in vivo prenatal gene transfer. Although survival remained limited due to the limitations of this mouse model, this study supports the potential for treatment of JEB by prenatal gene transfer.

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.

Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson's disease.

The ideal gene therapy for metabolical liver disorders would target hepatocytes before the onset of disease and be durable, non-toxic and non-immunogenic. Early gestational gene transfer can achieve such goals. Here, we demonstrate that prenatal gene transfer of human Atp7b reduces liver pathology and improves biochemical markers in Atp7b(-/-) mice, a murine model of Wilson's disease (WD). Following prenatal injection of lentivirus vector containing the human Atp7b gene under the transcriptional control of a liver-specific promoter, the full-length ATP7B was detectable in mouse livers for the entire duration of experiments (20 weeks after birth). In contrast to a marked pathology in non-injected animals, livers from age-matched treated mice consistently demonstrated normal gross and histological morphology. Hepatic copper content was decreased in the majority of treated mice, although remaining copper levels varied. Improvement of hepatic copper metabolism was further apparent from the presence of copper-bound ceruloplasmin in the sera and normalization of the mRNA levels for HMG CoA-reductase. With this approach, the complete loss of copper transport function can be ameliorated, as evident from phenotypical improvement in treated Atp7b(-/-) mice. This study provides proof of principle for in utero gene therapy in WD and other liver-based enzyme deficiencies.

Early gestational gene transfer of IL-10 by systemic administration of lentiviral vector can prevent arthritis in a murine model.

Gene therapy has been applied to murine models of rheumatoid arthritis (RA) using a number of different strategies to downregulate inflammation in synovial joints. However, prolonged joint expression has been problematic. Our laboratory has found that early gestational intravascular injection of lentiviral vector leads to efficient transduction and sustained transgene expression in articular cartilage and synovium. In this study, we show that in utero gene transfer of IL-10 can prevent and decrease pathology in a murine model of RA. Following prenatal injection of lentiviral vector containing murine IL-10 gene, the cytokine was detectable in the serum, and the green fluorescent protein reporter gene was detectable in chondrocytes and synoviocytes of adult mice up to 21 weeks of age. Adult mice that had been treated prenatally were later immunized against type II collagen to induce an autoimmune arthritis. Compared with controls, prenatally treated mice demonstrated delayed onset of arthritis, decreased frequency of arthritis and markedly decreased severity of disease, by both clinical and histological criteria. This effect was directly related to levels of IL-10 expression, but no immunosuppressive effects of the therapy were observed. This study demonstrates proof of principle for the prenatal prevention and amelioration of RA by early gestational gene transfer of the anti-inflammatory cytokine, IL-10.

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors.

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have recently shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this study we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8-10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

SLAM-enriched hematopoietic stem cells maintain long-term repopulating capacity after lentiviral transduction using an abbreviated protocol.

Gene transfer to long-term repopulating hematopoietic stem cells (HSCs) using integrating viral vectors is an important goal in gene therapy. The SLAM (signaling lymphocyte activation molecule)-family receptors have recently been used for the isolation of highly enriched murine HSCs. This HSC enrichment protocol is relatively simple, and results in an HSC population with comparable repopulating capacity to c-kit(+)lin(-)Sca-1(+) (KSL) HSCs. The capacity to withstand genetic manipulation and, most importantly, to maintain long-term repopulating capacity of SLAM-enriched HSC populations has not been reported. In this study, SLAM-enriched HSCs were assessed for transduction efficiency and in vivo long-term repopulating capacity after lentiviral transduction using an abbreviated transduction protocol and KSL-enriched HSCs as a reference population. SLAM- and KSL-enriched HSCs were efficiently transduced by lentiviral vector using a simple protocol that involves minimal in vitro manipulation and no pre-stimulation. SLAM-HSCs are at least equal to KSL-HSCs with respect to efficiency of transduction and maintenance of long-term repopulating capacity. Although there was a reduction in repopulating capacity related to enrichment and culture manipulations relative to freshly isolated bone marrow (BM) cells, no detrimental effects were identified on long-term competitive capacity related to transduction, as transduced cells maintained stable levels of chimerism in competition with non-transduced cells and freshly isolated BM cells. These results support the SLAM-HSC enrichment protocol as a simple and efficient method for HSC enrichment for gene transfer studies.

Biology of E1-deleted adenovirus vectors in nonhuman primate muscle.

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

Regulated gene expression in gene therapy.

The original model of gene therapy, that of efficient delivery, durable transfer, and stable expression of transgenes to correct a gene defect underlying an inherited disease, is limited in light of improved understanding of the processes involved. Techniques that enable regulated expression of transgenes may enhance safety and allow us to regulate the timing and level of expression with a goal of precisely targeting a therapeutic level between the extremes of suboptimal and supraoptimal thresholds. Using regulated systems to control protein expression has practical and possibly essential roles for the success of safe and effective gene therapy in a number of clinical situations. Pharmacologically regulated gene expression is an evolving tool, and no individual system may be effective in all clinical applications.

A quantitative nonimmunogenic transgene product for evaluating vectors in nonhuman primates.

The success of gene therapy depends on safe, effective vectors to transfer genetic information. We have developed a means to quantitatively assess efficacy of gene transfer vectors by using a biologically inert, secreted reporter molecule, the beta chain of chorionic gonadotropin (beta-CG). Using an isogenic beta chain subunit of CG in a recombinant adeno-associated virus (rAAV) vector, overall gene transfer of rhesus macaque muscle is demonstrated over time by measuring the serum concentration of beta-CG. Endogenous levels of gonadotropins are not detectable in healthy, nonpregnant primates as confirmed by the inability to detect serum levels of beta-CG prior to vector administration. The serum concentration of beta-CG also provides a measure for the transfer efficiency in liver and/or muscle in immunodeficient mice using recombinant adenovirus and rAAV vectors. No biological effect was observed in animals tested. Assaying for the serum level of beta-CG serves as a surrogate quantitative marker of gene transfer without interfering with the biology of the host or transduction process.

Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle.

Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.

Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus.

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.

Evidence that replication of human neurotropic JC virus DNA in glial cells is regulated by the sequence-specific single-stranded DNA-binding protein Pur alpha.

Initiation of polyomavirus DNA replication in eukaryotic cells requires the participation of the viral early protein T antigen, cellular replication factors, and DNA polymerases. The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised individuals. This virus exhibits a narrow host range and a tissue specificity that restricts its replication to glial cells of the central nervous system. Restriction of viral DNA replication due to species specificity of the DNA polymerase, coupled with glial cell-specific transcription of the viral early promoter, is thought to account for the brain-specific replication of JCV. In this report we demonstrate that overexpression of Pur alpha, a protein which binds to single-stranded DNA in a sequence-specific manner, suppresses replication of JCV DNA in glial cells. Results from footprinting studies indicate that Pur alpha and T antigen share a common binding region spanning the single-stranded ori sequence of JCV. Further, T antigen was capable of stimulating the association of Pur alpha with the ori sequence in a band shift assay. Whereas no evidence for simultaneous binding of Pur alpha and T antigen to single-stranded DNA has been observed, results from coimmunoprecipitation and Western blot (immunoblot) analyses of proteins derived from cells producing JCV T antigen indicate a molecular association of JCV T antigen and Pur alpha. The binding of Pur alpha to the single-stranded ori sequence and its association with T antigen suggest that Pur alpha interferes with the activity of T antigen and/or other regulatory proteins to exert its negative effect on JCV DNA replication. The importance of these findings in the reactivation of JCV in the latently infected individual under immunosuppressed conditions is discussed.

Formation of undifferentiated mesenteric tumors in transgenic mice expressing human neurotropic polymavirus early protein.

The human polyomavirus, JCV, is the established etiologic agent of the human demyelinating disease, progressive multifocal leukoencephalopathy (PML) seen in immunosuppressed individuals. In PML patients, the viral early protein, which is produced exclusively in glial cells is responsible for initiation of the viral lytic cycle. The JCV early protein, T-antigen, has greater than 70% homology to the well characterized SV40 early protein which has established oncogenic properties. To investigate the role of JCV T-antigen in tumorigenesis, transgenic mice containing the viral early genome were produced. Of the four positive transgenic animals, one developed severe neurological abnormalities and succumbed to death at 3 weeks of age. Another animal died with no visible gross pathology and the cause of death was not determined. The remaining two founders developed massive, undifferentiated, solid mesenteric tumors with no obvious neurological symptoms. Results from histologic analysis demonstrated the presence of highly cellular, poorly differentiated neoplastic cells in the tumor tissue. Electron microscopic evaluation of the tumor revealed the presence of a small blue cell-like tumor of epithelial/neuroectodermal origin. Results from RNA analysis by non-quantitative and highly sensitive RT-PCR indicated the presence of the JCV early transcript in various tissues, including kidney, liver, spleen, heart, lung, and brain, as well as in the tumors. However, analysis of the viral early protein by Western blot and immunohistochemistry indicated high level production of JCV early protein in the tumor tissue, but not in any other tissues. These observations present the first evidence for the development of inheritable neuroectodermal tumors induced by the human polyomavirus, JCV, early protein in a whole animal system.

Transcription of the JC virus archetype late genome: importance of the kappa B and the 23-base-pair motifs in late promoter activity in glial cells.

The transcription control region of the archetype strain of the human polyomavirus JC virus (JCV(Cy)), unlike its neurotropic counterpart (JCV(Mad-1)), contains only one copy of the 98-bp enhancer/promoter repeat with the 23-bp and the 66-bp insertion blocks. Early studies by us and others have indicated that the structural organization of JCV(Mad-1) is critical for glial cell-specific transcription of the viral genome. In addition, the kappa B regulatory motif found in the JCV(Mad-1) genome, which also exists in JCV(Cy), confers inducibility to the JCV(Mad-1) early and late promoters in response to extracellular stimuli. In this study, we have investigated the regulatory role of the 23- and the 66-bp blocks and their functional relationship to the kappa B motif in stimulating transcription of the Cy early and late promoters in glial cells. We demonstrate that mutations in the kappa B motif reduce the basal activity of the Cy early promoter and decrease the levels of its induction by phorbol myristate acetate or factors derived from activated T cells. Under similar circumstances, mutation in the kappa B motif completely abrogated the basal and the induced levels of transcription of the viral late promoter. Using deletion and hybrid promoter constructs, we have demonstrated that the 23-bp block of the Cy promoter plays a critical role in the observed inactivation of Cy late promoter transcription in glial cells. Results from DNA binding studies have indicated the formation of a common nucleoprotein complex with the 23-bp sequence, mutant kappa B (kappa B(mut)), and wild-type kappa B (kappa B(wt)). Analysis of this complex by UV cross-linking has identified a 40-kDa protein which binds to the 23-bp sequence and the kappa B motif. The importance of these findings for the activation of JCV(Cy) under various physiological conditions is discussed.

Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors.

The epidermal growth factor receptor has received much interest as a target for various antineoplastic agents, but a complication is that many normal tissues also express this receptor. We have previously identified in human glial tumors an 801-bp in-frame deletion within the epidermal growth factor receptor gene that created a novel epitope at the junction. By using Western blot assays with a mutant-specific antibody as a rapid and sensitive means for detecting this alteration in primary human tumors, it was found that 57% (26 of 46) of high-grade and 86% (6 of 7) of low-grade glial tumors, but not normal brain, express this protein. This altered receptor was also present in 66% (4 of 6) of pediatric gliomas and 86% (6 of 7) of medulloblastomas, 78% (21 of 27) of breast carcinomas, and 73% (24 of 32) of ovarian carcinomas. The fact that this receptor is frequently found in tumors but not in normal tissue makes it an attractive candidate for various antitumor strategies.

Isolation and characterization of a type II JC virus from a brain biopsy of a patient with PML.

Brain tissue of a patient with multiple myeloma suffering from neurological disorders similar to those seen in progressive multifocal leukoencephalopathy (PML) patients was evaluated for the presence of the papovavirus, JCV. Results from polymerase chain reaction (PCR) revealed the presence of JCV with structural organization at the control region which is distinct from well-characterized isolates, ie Mad-1 and Mad-4. The control region of the new isolate, named JCVPhila-1' contains a 22 nucleotide insertion which separates the TATA box from the NF-1 regulatory motif. Only 18 nucleotides of the insert are duplicated in the second copy of the enhancer/promoter of the new isolate, which is 84 nucleotides in size. Results from a transcription assay indicate a modest elevated level of JCVPhila-1 early promoter activity compared to that of JCVMad-4 in glial cell lines. The basal and T-antigen-induced transcriptional activities of the JCVPhila-1 late promoter was lower with respect to Mad-4 late gene activity in glial cells. Of particular interest was the observation that in the cells producing the early protein, T-antigen, JCVPhila-1 DNA replicated more efficiently then the Mad-4 DNA. These results suggest that the alterations seen in the JCVPhila-1 control region may differentially influence early and late gene expression and facilitate amplification of the viral genome in cells derived from the CNS.

The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo.

The role of the insulin-like growth factor I receptor (IGF-IR) in programmed cell death has been investigated in vivo in a biodiffusion chamber, where the extent of cell death could be determined quantitatively. We found that a decrease in the number of IGF-IRs causes massive apoptosis in vivo in several transplantable tumors, either from humans or rodents. Conversely, an overexpressed IGF-IR protects cells from apoptosis in vivo. We also show that the same conditions that in vitro cause only partial growth arrest with a minimum of cell death, induce in vivo almost complete cell death. We conclude that the IGF-IR activated by its ligands plays a very important protective role in programmed cell death, and that its protective action is even more striking in vivo than in vitro.