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Biomedical subjects

M A Kay

Publications and source records attributed to M A Kay.

At least 19 recordsLinked to original sources
PubMed2003

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer.

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

Animals↗
PubMed2003

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy.

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

Animals↗
PubMed2001

Approaches for generating recombinant adenovirus vectors.

Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction. These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions. By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism. In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.

Adenoviridae↗
PubMed2001

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Adenoviridae↗
PubMed2001

Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver.

The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human alpha1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 microg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.

Animals↗
PubMed2001

Epstein-Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice.

We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.

Animals↗
PubMed2001

Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo.

Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

Animals↗
PubMed2001

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Animals↗
PubMed2001

Hepatocyte transplantation: clinical and experimental application.

Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Based on a significant body of work, the technique of hepatocyte transplantation has recently moved into the clinic in order to reestablish liver function without organ transplantation or to bridge the time between whole-organ liver transplantation. In addition, hepatocyte transplantation has also been proposed as a liver-directed gene therapy for a number of inherited hepatic disorders by transplanting either freshly isolated hepatocytes or genetically altered hepatocytes. To establish a research system based on the developing technology of hepatocyte transplantation, chimeric small animal models using human hepatocytes have recently been established, which would allow the study of human hepatocyte-specific functions, such as hepatitis viral infection and replication in vivo. Various aspects related to the recent progress and existing obstacles in the area of hepatocyte transplantation are summarized in this report.

Animals↗
PubMed2001

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.

Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.

Genetic Therapy↗
PubMed2001

A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

Adenoviridae↗
PubMed2001

Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.

Animals↗
PubMed2000

Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver.

Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. The present study used human immunodeficiency virus (HIV)-based lentiviral vectors containing an EF1alpha enhancer/promoter driving human factors VIII (hFVIII) or IX (hFIX) complementary DNA expression for portal vein injection into C57Bl/6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 50-60 ng/mL. Partial hepatectomy resulted in a 4- to 6-fold increase (P < 0.005) in serum hFIX of up to 350 ng/mL compared with the nonhepatectomized counterparts. The expression of plasma hFVIII reached 30 ng/mL (15% of normal) but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII antibodies. However, hFVIII levels were persistent in immunodeficient C57Bl/6 scid mice, suggesting humoral immunity-limited gene expression in immunocompetent mice. This study demonstrates that lentiviral vectors can produce therapeutic levels of coagulation factors in vivo, which can be enhanced with hepatocellular proliferation.

Animals↗
PubMed2000

Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro.

We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.

3' Untranslated Regions↗
PubMed2000

Efficient lentiviral transduction of liver requires cell cycling in vivo.

Human-immunodeficiency-virus (HIV)-based lentiviral vectors are a promising tool for in vivo gene therapy. Unlike Moloney-murine-leukaemia-based retroviruses (MLV), lentiviruses are believed to stably transduce quiescent (non-cycling) cells in various organs. No previous studies, however, have directly established the cell-cycle status of any transduced cell type at the time of vector administration in vivo. In vitro studies using wild-type HIV or HIV-based vectors have shown that, in some cases, cell-cycle activation is required for infection, even though cellular mitosis is not an absolute requirement for integration. Even if the block in reverse transcription is overcome in quiescent T cells, productive infection by HIV cannot be rescued in the absence of cell-cycle activation. The potential use of these vectors for gene therapy prompted our study, which establishes a cell-cycle requirement for efficient transduction of hepatocytes in vivo.

Animals↗
PubMed2000

Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses.

Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

Animals↗