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Biomedical subjects

Christopher E Walsh

Publications and source records attributed to Christopher E Walsh.

16 recordsLinked to original sources
PubMed2006

Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats.

BACKGROUND: Intrathecal (IT) gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks) impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as > or = 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF) modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV), one of the most promising vector types for clinical use. RESULTS: Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV) vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 x 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP) for > or = 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. CONCLUSION: sc-rAAVl mediates stable IT transgene expression for > or = 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2. Experiments presented here will provide a rational basis for utilizing IT rAAV gene transfer in basic and translational studies on chronic pain.

Animals↗
PubMed2006

RNA repair for haemophilia A.

The mainstay of gene transfer studies is the use of wild-type cDNAs to effect phenotypic correction of diseases. However, this strategy is not feasible for genetic diseases caused either by mutations of large genes or by dominant-negative mutations, or where the regulation of the gene is critical. In this review, we will discuss a novel RNA reprogramming strategy - spliceosome-mediated RNA trans-splicing - where the pre-messenger RNA is modified by the splicing of two independent RNA species. The use of trans-splicing to effect phenotypic change in the hereditary bleeding disorder haemophilia A will be discussed.

Animals↗
PubMed2005

Spliceosome-mediated RNA trans-splicing.

RNA repair or reprogramming is a new avenue for human gene therapy. Unlike conventional gene therapy, in which exogenous cDNAs are introduced into cells, RNA repair approaches, which are based on spliceosome-mediated pre-mRNA trans-splicing, trans-splicing ribozymes, and tRNA-splicing endonuclease, allow the correction of endogenous RNA species. Recently published accounts that in vivo phenotypic correction of a variety of inherited diseases can be achieved by RNA repair are encouraging. Nevertheless, the science of RNA repair for treatment of human diseases is just beginning and faces several scientific and technical challenges that must be addressed and surmounted. In this review, we summarize recent advances in spliceosome-mediated pre-mRNA trans-splicing. We also provide an update on the progress of this emerging technology toward the development of molecular therapy and diagnosis for human diseases and discuss the outstanding issues and challenges confronting RNA therapeutics.

Animals↗
PubMed2005

Intrathecal gene transfer by adeno-associated virus for pain.

Chronic pain is among the most prevalent medical problems, affecting more than half of patients with advanced cancer and many with other common diseases. Current analgesics often fail to provide satisfactory symptom relief and frequently cause severe side effects. Intrathecal (IT) gene transfer is an attractive method for pain research in rodent models, because it allows targeting of a wide variety of secretable peptides and proteins to the spinal cord, an important neural center for the processing of nociceptive signals. The potential of IT gene transfer for improving opioid therapy and for validating new analgesic targets, such as cytokines involved in spinal glial activation, is discussed. The IT space has been notoriously resistant to efficient gene transfer, limiting therapeutic gene expression to less than 2 weeks with most vector systems. Recent progress with adeno-associated virus (AAV) technology allowed efficient long-term gene expression, facilitating studies reflective of the chronic nature of many pain states. AAV is one of the most advanced gene therapy vectors currently undergoing clinical trials for a variety of disorders. In patients, AAV vectors could be administered intrathecally by a lumbar puncture, a safe procedure routinely performed at the bedside. AAV vectors may therefore become an important tool for translational studies to validate newly identified therapeutic targets in clinical pain states.

Analgesics, Opioid↗
PubMed2004

New paradigms for gene transfer: RNA trans-splicing and small interfering RNA as therapeutic strategies.

If successful, the sustained and regulated expression of therapeutic proteins secreted from a variety of tissues would revolutionize the medical treatment of hematologic diseases. The current paradigm that has dominated the gene therapy field since its inception has been the transfer of complementary DNAs (cDNAs) that encode for therapeutic proteins. The transfer of cDNAs can only correct autosomal recessive and sex-linked disorders. In most cases, cDNAs are constructed that lack their endogenous regulatory elements and therefore lose their intrinsic regulation of gene expression. In this article we will describe the use of RNA species to either suppress unwanted gene activity or to repair defective genes. Examples of RNA inhibition and repair will be discussed.

Gene Transfer Techniques↗
PubMed2004

AAV vectors for hemophilia B gene therapy.

Adeno-associated viral (AAV) vector is attracting significant interest for use in gene therapy for genetic diseases, because of its unique and advantageous characteristics, compared to other currently available viral vectors. Eight natural serotypes of AAV have been identified, of which AAV serotype 2 is the one best characterized and most widely used in current gene delivery studies. The application of AAV serotype 2 in hemophilia B gene therapy is a promising development in gene therapy for genetic diseases such as hemophilia. Preliminary studies have demonstrated relation and distinction of host, genome sequences, replication, tropism, packaging of recombinant virions and cross-reactivity of neutralizing antibodies among different serotypes of AAV. This review summarizes the progress of studies in AAV serotypes and pertinent applications in hemophilia B gene therapy. The latest progress in gene delivery of coagulant factor IX (for hemophilia B) using AAV serotype vectors is described in detail.

Adenoviridae↗
PubMed2003

Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing.

Conventional gene therapy of hemophilia A relies on the transfer of factor VIII (FVIII; encoded by the F8 gene) cDNA. We carried out spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutant FVIII mRNA. A pre-trans-splicing molecule (PTM) corrected endogenous FVIII mRNA in F8 knockout mice with the hemophilia A phenotype, producing sufficient functional FVIII to correct the hemophilia A phenotype. This is the first description of phenotypic correction of a genetic defect by RNA repair in a knockout animal model. Our results indicate the feasibility of using SMaRT to repair RNA for the treatment of genetic diseases.

Animals↗
PubMed2003

Phenotype correction of Fanconi anemia group A hematopoietic stem cells using lentiviral vector.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure due to defective stem cell function. FA patients' cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC), exposure to which results in cytogenetic aberrations and cell death. To date Moloney murine leukemia virus vectors have been used in clinical gene therapy. Recently, third-generation lentiviral vectors based on the HIV-1 genome have been developed for efficient gene transfer to hematopoietic stem cells. We generated a self-inactivating lentiviral vector expressing the FA group A cDNA driven by the murine stem cell virus U3 LTR promoter and used the vector to transduce side-population (SP) cells isolated from bone marrow of Fanconi anemia group A (Fanca) knockout mice. One thousand transduced SP cells reconstituted the bone marrow of sublethally irradiated Fanca recipient mice. Phenotype correction was demonstrated by stable hematopoiesis following MMC challenge. Using real-time PCR, one proviral vector DNA copy per cell was detected in all lineage-committed cells in the peripheral blood of both primary and secondary recipients. Our results suggest that the lentiviral vector transduces stem cells capable of self-renewal and long-term hematopoiesis in vivo and is potentially useful for clinical gene therapy of FA hematopoietic cells.

Animals↗
PubMed2003

Gene therapy progress and prospects: gene therapy for the hemophilias.

Recent gene transfer trials for hemophilia A and B, bleeding disorders lacking either functional factor VIII or IX, respectively, have produced tantalizing results, suggesting that the potential to correct these bleeding disorders at a molecular level may be at hand. Genetic correction of the hemophilias represents a model system to develop a basic understanding of how gene therapy will be achieved. The goals for hemophilia gene transfer require the long-term therapeutic production of the coagulant protein without stimulating an immune response to the transgene product or the vector. Based on a scientific understanding of the molecular and cellular defects, leading to the bleeding phenotype, impressive strides have been made in the last 2 years.

Gene Transfer Techniques↗
PubMed2003

Congenital bleeding disorders.

Both clinical and basic problems related to the congenital bleeding disorders continue to confront hematologists. On the forefront are efforts to bring genetic correction of the more common bleeding disorders such as hemophilia A to the clinic in a safe and accessible manner. A second issue, particularly for patients with hemophilia, is the development of inhibitors-questions of how they arise and how to prevent and treat these problems that confound otherwise very successful replacement therapy and allow patients to maintain normal lifestyles. A third issue is the continuing question of diagnosis and management of von Willebrand disease, the most common congenital bleeding disorder, especially in individuals who have borderline laboratory values, but have a history of clinical bleeding. In Section I, Dr. Christopher Walsh discusses general principles of effective gene transfer for the hemophilias, specific information about viral vectors and non-viral gene transfer, and alternative target tissues for factor VIII and factor IX production. He highlights information about the immune response to gene transfer and reviews data from the hemophilia gene transfer trials to date. The future prospects for newer methods of therapy such as RNA repair and the use of gene-modified circulating endothelial progenitors are presented as possible alternatives to the more traditional gene therapy approaches. In Section II, Dr. Nigel Key focuses on inhibitor development in patients with hemophilia A. He reviews the progress in our understanding of the risk factors and presents newer information about the immunobiology of inhibitor development. He discusses the natural history of these inhibitors and the screening, laboratory diagnosis, and treatment, including the use of different modalities for the treatment of acute bleeding episodes. Dr. Key also presents information about the eradication of inhibitors by immune tolerance induction and reviews recent information from the international registries regarding the status and success of immune tolerance induction. In Section III, Dr. Margaret Rick discusses the diagnosis, classification, and management of von Willebrand disease. Attention is given to the difficulty of diagnosis in patients with mild bleeding histories and borderline laboratory test results for von Willebrand factor. She presents the value of different laboratory assays for both diagnosis and classification, and she relates the classification of von Willebrand disease to the choice of treatment and to the known genetic mutations. Practical issues of diagnosis and treatment, including clinical cases, will be presented.

Blood Coagulation Disorders↗
PubMed2003

Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer.

Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells.

Animals↗
PubMed2002

Expression of human factor VIII by splicing between dimerized AAV vectors.

Adeno-associated virus (AAV) is a useful vector for hemophilia gene therapy, but the limited effective packaging capacity of AAV (5 kb) appears to be incompatible with factor VIII (gene symbol F8) cDNA (7 kb). Although we previously demonstrated efficient packaging and expression of B-domain-deleted human F8 (BDD-F8) using a single AAV vector, the packaging limit still excludes the use of large/strong regulatory elements. Here we exploited the split AAV vector technology that expands the packaging capacity of AAV through head-to-tail dimerization. To test the feasibility of AAV heterodimerization for F8 expression, we generated a 5' vector that includes a large enhancer/promoter cassette linked with exons 1-12 of the F8 cDNA and a half-intron-carrying splice donor site. A complementing 3' vector contains another half-intron-carrying splice acceptor site linked with the remaining F8 cDNA and a polyadenylation signal. Following coinfection of 293 and HepG2 cells, the 5' and 3' vectors together produced functional human factor VIII protein at a level of 120 mU/ml (24 ng/ml). No factor VIII protein was detected if only one of the vectors was used. Correct head-to-tail vector dimerization as well as spliced BDD-F8 mRNA was detected by DNA PCR and RT-PCR, respectively. Furthermore, intraportal injection of two rAAV/F8 vectors in immunodeficient mice produced 2% of the normal level of factor VIII for four months. Our results demonstrate the potential use of AAV dimerization for F8 expression.

Animals↗
PubMed2002

Gene therapy for the hemophilias.

Recent advances in the field of gene transfer are producing tantalizing results suggesting that the potential to correct disease at a molecular level may be at hand. Genetic correction of the hemophilias--bleeding disorders that stem from the deficiency of functional factor VIII or IX--represent models for the development of a basic understanding of how gene therapy will be achieved. The goals for hemophilia gene transfer are to produce therapeutic amounts of the coagulant protein while minimizing an immune response or antibody inhibitor. This requires the use of nontoxic vectors to deliver genes that express the protein in a functional form for the life of the patient. Based on a scientific understanding of the molecular and cellular defects leading to the bleeding phenotype, gene transfer studies at the laboratory and clinic have produced exciting results. The author here provides a critical assessment of the state of hemophilia gene transfer and its relevance to the field as a whole.

Antibody Formation↗
PubMed2002

Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides.

Correct human beta-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with beta-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/beta(E) genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in beta-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human beta-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.

Animals↗
PubMed2002

Hemophilia gene therapy: novel rAAV vectors and RNA repair strategy.

Hemophilia results from a deficiency of coagulation Factor VIII or IX and manifests clinically as spontaneous bleeding into the large joints and soft tissue. Current treatment relies on the intravenous infusion of recombinant or purified Factor proteins. Factor infusion is effective, but transient due to the short half-life of Factor proteins. Recent developments in gene transfer technology have led to new strategies using molecular therapeutics as permanent treatment for bleeding disorders. This review describes recent novel molecular strategies for the treatment of the hemophilias.

Dependovirus↗