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M A Kay

Publications and source records attributed to M A Kay.

At least 91 records · Page 5Linked to original sources
PubMed1995

Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration.

Recombinant adenovirus vectors are efficient at transferring genes into somatic tissues but are limited for use in clinical gene therapy by immunologic factors that result in the rapid loss of gene expression and inhibit secondary gene transfer. This study demonstrates that systemic coadministration of recombinant adenovirus with soluble CTLA4Ig, which is known to block co-stimulatory signals between T cells and antigen presenting cells, leads to persistent adenoviral gene expression in mice without long-term immunosuppression. This form of immunotherapy greatly enhances the likelihood that recombinant adenovirus vectors will be useful for human gene therapy.

Abatacept↗
PubMed1995

Adenovirus-mediated transfer of the amphotropic retrovirus receptor cDNA increases retroviral transduction in cultured cells.

The presence of functional amphotropic receptors on the cell surface is necessary for amphotropic retrovirus-mediated gene transfer. A recombinant adenoviral vector that expresses the receptor for amphotropic retrovirus (RAM) was constructed and used to express the receptor cDNA in different cell types in culture. Transfer of the RAM cDNA increased amphotropic retroviral-mediated transfer from 0 to 60% in Chinese hamster ovary cells. RAM expression increased retroviral transduction four- to eight-fold from 2-4% to 18%-35% in HeLa, Namalva, and X63 cells, but had no effect on 208F and HepG2 cells which have high baseline retroviral transduction rates of about 50%. For the purpose of application to ex vivo gene therapy, primary mouse hepatocytes were studied in a similar manner. Hepatocytes had a baseline transduction efficiency of about 40% and did not have increased rates of retroviral-mediated gene transfer with expression of recombinant RAM. This recombinant adenoviral vector conferred infection of amphotropic retrovirus into cells that were relatively resistant to infection, thus offering a rapid and easy method to stably introduce genes into these cell lines.

Adenoviridae↗
PubMed1995

A modified urokinase plasminogen activator induces liver regeneration without bleeding.

Direct retrovirus-mediated hepatic gene transfer results in permanent gene expression; however, gene transfer requires surgical hepatectomy (to stimulate cell division) and has been inefficient. We recently used recombinant adenovirus vectors that transiently expressed urokinase from mouse hepatocytes to induce hepatocellular regeneration in place of a partial hepatectomy. The adenovirus method allowed for five-fold more efficient retrovirus transduction in vivo compared to the conventional partial hepatectomy approach. The major problem with the urokinase-mediated hepatic regeneration was the transient secretion of urokinase into the bloodstream that led to hypocoagulation. To circumvent this side-effect, the urokinase protein was modified by adding amino-terminal and carboxy-terminal endoplasmic reticulum retention signals. The recombinant urokinase molecules expressed from adenoviral vectors remained in hepatocytes, were enzymatically active, and resulted in similar rates of hepatic regeneration as found with the secreted urokinase. Modified urokinase-mediated liver regeneration was equally capable of allowing retrovirus-mediated gene transfer in vivo. Thus, the method of direct retrovirus transduction of hepatocytes becomes clinically relevant as the technology becomes safer.

Adenoviridae↗
PubMed1995

Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

Adenoviridae↗
PubMed1995

Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus-polylysine-DNA complexes.

The E1 deleted adenoviral vectors are efficient at gene transfer to cells in culture or in animals. However, their use is limited because of an immune-mediated loss of transduced cells. This immune response is believed to result from low-level production of viral antigens from these vectors after gene transfer. The early region 4 (E4) of adenovirus produces a number of proteins that play an important role in adenoviral and host gene regulation during infection of mammalian cells. There is interest in developing E4 deficient adenovirus for gene therapy, especially in the context of developing a combined E1/E4 deleted vector. Towards this goal, a method by which to complement and propagate an E4 deficient adenovirus (dl 1014) in the E1 complementing 293 cell line, using a novel and simple rescue technique, has been developed. Purified adenovirus deficient in E4 gene expression (dl 1014) was conjugated to expression plasmids containing the E4-open reading frame 6 gene or complete E4 region to produce adenovirus-polylysine-DNA complexes that were used to transfect 293 cells. The derived virus obtained from this transfection did not replicate on 293 cells but did replicate on W162 cells (E4+) confirming that the virus was indeed deleted for E4. Viral yield was high ranging from 3 x 10(8) to 9 x 10(8) plaque forming units per 10(6) 293 cells. This method has general application to the production of new adenoviral mutants that will be useful for developing second generation adenoviral vectors.

Adenovirus E4 Proteins↗
PubMed1995

Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient inbred strains.

High efficiency gene transfer and gene expression in hepatocytes in vivo can be achieved using recombinant adenoviral vectors. However, the persistence of gene expression in different experimental animal models has been variable. To determine if similar differences could be observed in a single species, persistence of gene expression was studied in inbred strains of mice using a recombinant adenoviral vector that expresses human alpha 1-antitrypsin. Marked variability in the persistence of gene expression ranging from several weeks (C3H/HeJ and Balb/c) to more than 3 months [C57Bl/6, B10.A(2R) and B10.BR] was observed when this vector was transduced in different strains of inbred mice. This variability did not correlate with H-2 type. To evaluate the role of T and B cell immunity in the persistence of gene expression, congenic C3H-scid and Balb/c-scid mice were studied and found to have indefinite gene expression from transduced hepatocytes. These animals unlike their immunocompetent counter-parts were able to undergo secondary transduction of hepatocytes with a different recombinant adenoviral vector. These findings suggest that as yet unidentified genetic loci influence the persistence of adenovirus-mediated hepatic gene expression in vivo, and these effects are mediated at least in part, by the antigen specific immune system.

Adenoviridae↗
PubMed1995

Therapeutic serum concentrations of human alpha-1-antitrypsin after adenoviral-mediated gene transfer into mouse hepatocytes.

Alpha-1-antitrypsin is a relatively common genetic deficiency that results in early emphysema. The liver as the natural source of most alpha-1-antitrypsin synthesis was the target organ selected for gene replacement therapy studies. Previous work used recombinant retroviral vectors that encode the human alpha-1-antitrypsin cDNA for ex vivo and direct in vivo transduction of hepatocytes in dogs and rodents. This approach led to low levels of human protein in the serum of recipients. In this study, recombinant adenoviral vectors that express the human alpha-1-antitrypsin cDNA under the transcriptional control of the phosphoglycerate kinase (PGK) or RSV-LTR promoters have been constructed and used for the direct transduction of mouse hepatocytes in vivo. The animals transduced with the recombinant adenoviral vectors had therapeutic serum levels of human alpha-1-antitrypsin of up to 700 micrograms/mL. Thus, adenovirus-mediated gene transfer of the hAAT cDNA into the liver was able to produce therapeutic serum concentrations of protein.

Adenoviridae↗
PubMed1994

In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.

Adenoviridae↗
PubMed1994

Gene therapy for metabolic disorders.

Most diseases caused by genetic deficiencies could, in theory, be treated by the introduction and expression of a normal gene into an appropriate target tissue. It seems likely that gene therapy strategies for most metabolic disorders will not require strict gene regulation, as a fraction of the normal levels of gene activity could result in amelioration or significant improvement in the clinical outcome. Gene therapy is making rapid progress towards the goal of treating various disorders: here, we summarize the state of gene therapy for metabolic disorders.

Animals↗
PubMed1993

Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies.

Adenoviridae↗
PubMed1993

In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs.

The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

Animals↗
PubMed1993

Hepatic gene therapy: efficient retroviral-mediated gene transfer into rat hepatocytes in vivo.

The rat is an excellent model for gene therapy because there are many rat models for human diseases. We have developed a simple and efficient method to deliver genes to the rat liver using recombinant retroviral vectors. A 70% partial hepatectomy followed by retroviral infusion into the portal vein results in 10-15% hepatocyte transduction in vivo. This is 10 times more efficient than in the mouse due partially to the observation that the rat livers have much more synchronous hepatocyte replication after partial hepatectomy. Using a recombinant retroviral vector containing the human alpha 1-antitrypsin cDNA, persistent expression of the human protein in recipient rat plasma was observed for at least six months and at a level that is 10 times greater than the mouse. Thus, rats can serve as an excellent model for gene therapy of metabolic disorders secondary to hepatic deficiencies.

Animals↗
PubMed1993

Fallen fontanelle: culture-bound or cross-cultural?

Rather than bound to one culture, fallen fontanelle has been labeled as an illness or recognized as a symptom through time and space. The condition may be called siriasus, sitibundum, fontanellae collapsus, el apostema cálido del cerebro, Blatfallen, Blattschiessen, entzündung des Gehirns und der Gehirnhäute der Kleinen Kinder, coup de soleil, sorte de maladie causée par l'inflammation des membranes du cerveau, head-mould-shot, mollera caída, desmollerado, gual, split skull, sutt, nhova, kubabula, chipande, phogwana and dehydration. Defining features of this condition as well as prevention and treatment have corresponded to the specific cultural setting and ethnographic present. Fallen fontanelle (or fontanel) is "a culturally interpreted symptom rather than culture-bound" (Low 1985). The methodological perspective is an ethnohistorical recounting of change in the meaning of this symptom.

Central America↗
PubMed1993

Assessment of recombinant adenoviral vectors for hepatic gene therapy.

Recombinant adenoviral vectors have recently been used to transfer genes into a number of different cell types in vitro and in vivo. A recombinant adenoviral vector bearing the Escherichia coli beta-galactosidase (beta-gal) gene was used to quantitate the frequency of hepatocyte transduction in the mouse after direct viral infusion into the portal vein. When 10(10) adenoviral particles were infused, over 95% of the hepatocytes were transduced in vivo as determined by x-gal staining. The transduction protocol is relatively safe in that there is no detectable helper virus production in transduced animals and that very few extrahepatic cells are transduced by this method. There is also no evidence of significant liver pathology unless substantially greater quantities of virus are used. However, the transduced hepatocytes do not appear to persist in vivo because the percentage of hepatocytes expressing beta-gal declined over time. Four months after the procedure, 0.5-10% of the hepatocytes contain detectable beta-gal activity in vivo. The change in beta-gal-positive cells correlates with decreasing amounts of adenoviral DNA. Thus, current recombinant adenoviral vectors may have clinical applications in gene therapy for acute hepatic disorders.

Adenoviruses, Human↗
PubMed1992

Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes.

The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 10(9) hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human alpha 1-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had significant human alpha 1-antitrypsin in the serum for 1 month. Although the serum levels of human alpha 1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.

Animals↗
PubMed1992

Reconstitution of enzymatic activity in hepatocytes of phenylalanine hydroxylase-deficient mice.

Phenylketonuria (PKU) is a metabolic disorder secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). The recent creation of a mouse strain for PAH deficiency has provided an excellent model system to explore the possibility of its phenotypic correction by hepatic gene therapy. A recombinant retrovirus containing the mouse PAH cDNA under the transcriptional control of the human CMV promoter was constructed and used to transduce hepatocytes isolated from PAH-deficient mice. Viral-transduced hepatocytes produced dramatically higher levels of mouse PAH mRNA as compared to control mock-infected hepatocytes. The PAH mRNA was translated efficiently into PAH protein that is capable of converting phenylalanine to tyrosine in vitro. These results demonstrate that the PAH-deficient mouse hepatocytes can be readily reconstituted by retroviral-mediated gene transduction, which is a crucial step towards somatic gene therapy for PKU.

Animals↗
PubMed1992

Human gene therapy: present and future.

The hematopoietic system and the liver are two primary target organs for attempting somatic gene therapy of hereditary deficiencies. Several leading laboratories have recently been able to demonstrate that bone marrow cells from rodents and non-human primates can be successfully transduced with foreign genes, resulting in the functional expression of these genes in culture. The genetically reconstituted cells can subsequently be transplanted into X-irradiated recipients, and expression of the transduced genes is observed in the recipients for more than 6 months. Subsequently, gene transfer into peripheral T-lymphocytes in humans has been attempted, and the clinical trials are currently in progress. The liver is the other major organ under intensive investigation. Primary hepatocytes can be isolated from rodents, rabbits, and dogs, and successfully transduced with recombinant retroviruses. After autologous transplantation, long term survival of the engrafted cells in vivo has been observed. More recently, it has been shown that human hepatocytes can also be efficiently transduced with recombinant retroviruses. These experimental results have laid the foundation for somatic gene therapy of hereditary deficiencies in humans in the future.

Animals↗