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Biomedical subjects

W F Goins

Publications and source records attributed to W F Goins.

At least 37 records · Page 2Linked to original sources
PubMed1997

Herpes simplex virus vectors for gene transfer to the nervous system.

Herpes simplex virus (HSV) represents a candidate gene transfer vector for the treatment of nervous system disease. It has many natural biological features which make it attractive for gene delivery to a variety of tissues. The virus naturally establishes a latency in sensory neurons of the peripheral nervous system, wherein the virus in maintained as an extrachromosomal DNA element in the absence of viral lytic gene expression without altering the metabolism of the host neuron. The virus possesses a neuronal latency-specific promoter system which remains active long-term, while other viral and cellular promoters are repressed. Replication defective virus recombinants have been engineered to delete multiple essential immediate early gene functions rendering these new mutants significantly less cytotoxic to neurons and other cells in culture. Further developments in regulating transgene expression and reducing virus toxicity will continue to aid the design and use of these vectors for therapeutic applications for the nervous system.

Animals↗
PubMed1996

Gene transfer to neurons using herpes simplex virus-based vectors.

One important outgrowth of molecular medicine is the development of technologies for the transfer of therapeutic genes to cells in culture and tissues in vivo, which promises to revolutionize both experimental biomedical science and the clinical practice of medicine. Fundamental obstacles must still be overcome to create safe and efficient gene delivery vectors specifically designed for individual tissue types, and special strategies will be required for direct in vivo gene transfer to neurons because these cells are postmitotic and cannot be removed for transduction. Herpes simplex virus type 1 (HSV-1), a neurotropic virus that naturally establishes a latent state in neurons, has many unique features that make it suitable as a gene transfer vector for the nervous system. In this review we describe the molecular biology of HSV-1, strategies for reducing potential pathogenesis of the recombinant vector, and methods for expressing transgenes from the vector genome. Gene transfer experiments using recombinant HSV-1-based vectors and defective HSV-1 vectors (amplicons) for gene transfer are also described and evaluated in terms of efficiency and safety.

Alzheimer Disease↗
PubMed1995

HSV as a gene transfer vector for the nervous system.

Gene therapy for diseases of the nervous system requires vectors capable of delivering the therapeutic gene into postmitotic cells in vivo. Herpes simplex virus type 1 is a neurotropic virus that naturally establishes latency in neurons of the peripheral nervous system. Replication defective HSV vectors have been developed; these are deleted for at least one essential immediate early regulatory gene, rendering the virus less cytotoxic, incapable of reactivation, but still capable of establishing latency. Foreign genes can be vigorously expressed from an HSV-based vector in a transient manner in brain and other tissues. Long-term but weak foreign gene expression may be achieved in the nervous system by exploiting the transcriptional control mechanisms of the natural viral latency active promoter. To meet the needs of specific applications, either highly active long-term or regulatable transgene expression will be needed, requiring further studies in order to design the appropriate latency-based promoter systems.

Gene Transfer Techniques↗
PubMed1995

Two herpes simplex virus type 1 latency-active promoters differ in their contributions to latency-associated transcript expression during lytic and latent infections.

Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.

Animals↗
PubMed1995

Herpes simplex virus type 1 vector mediated gene transfer to muscle.

Herpes simplex virus type 1 (HSV-1) has a broad host range although in natural human infections the virus is neurotropic, establishing latent infections in sensory neurons where the viral DNA persists as an intact episome. The establishment of latency does not depend on viral replication functions, suggesting that infection of non-neuronal cells, including tissue of myogenic origin, by replication defective mutants may result in genome persistence in a similar episomal state. In this report a replication defective HSV-1 recombinant vector containing the beta-galactosidase reporter gene under transcriptional control of the strong human cytomegalovirus immediate-early gene promoter (HCMV IEp-lacZ) was used to infect muscle cells in vitro and in vivo. This replication defective mutant virus (d120), deleted for both copies of the essential immediate-early gene (ICP4) and thus incapable of expressing early and late viral genes, displayed highly reduced cytotoxicity in myogenic cells. This vector infected both myoblasts and myotubes in culture with transgene expression persisting for at least 8 days. The transduction efficiency in myotubes was similar to myoblasts at several multiplicities of infection (MOIs), suggesting that HSV could infect differentiated muscle fibers and that myoblast differentiation would neither prevent expression of the cellular receptor(s) for the virus nor inhibit viral penetration. Direct inoculation of mouse muscle fibers in vivo with 10(6) to 10(8) plaque forming units (p.f.u.) of vector was sufficient to transduce significant numbers of muscle fibers in newborn mice and some fibers in adult normal and mdx mice. These results suggest that recombinant HSV-1 vectors may be useful for gene transfer to muscle.

Animals↗
PubMed1994

Gene transfer to brain using herpes simplex virus vectors.

Herpes simplex virus type 1 represents an ideal candidate for development as a vehicle for gene transfer to postmitotic neurons of the central nervous system. The natural biology of this virus makes it well suited for this purpose as it is capable of infecting a variety of neuronal cell types in the brain where the viral genome can persist indefinitely in a latent state. In latency, the viral lytic genes are transcriptionally silent and a unique set of latency-associated transcripts are expressed. Two impediments to using herpes simplex virus vectors must be overcome: (1) A noncytotoxic mutant virus backbone must be engineered, and (2) a suitable promoter-regulator that stably expresses foreign genes from the vector genome during latency must be constructed. Deletion of specific immediate early genes from the vector can render the virus nontoxic to neurons in culture and in vivo following stereotactic inoculation into specific regions of the brain. Because these viruses cannot replicate, they enter latency on infection of central nervous system neurons. A number of viral and cellular promoters have been tested for their ability to express genes during latency. Strong viral promoters and neurospecific promoters display transient activity. Although the promoter regions for the latency-associated transcripts are highly active in the peripheral nervous system, they show low-level but persistent activity in the brain. Experiments are in progress to exploit RNA polymerase III gene promoters or novel recombinant promoters capable of auto-inducing their own expression in order to increase gene expression during latency in brain neurons.

Animals↗
PubMed1994

A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats.

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.

Animals↗
PubMed1994

Herpes simplex virus vectors and gene transfer to brain.

Experiments are in progress to engineer herpes simplex virus type 1 as a gene transfer vector for the nervous system. This virus is well suited for this purpose due to its ability to persist for long periods in a latent state in neurons wherein lytic genes are inactive and a unique viral promoter is capable of inducing transcription from the latent viral genome. The virus can be highly cytotoxic for neurons, however, and thus genes which initiate the lytic cycle must be deleted from the genome in order to force the virus into latency. Candidate genes include the immediately early functions ICP0, ICP27, ICP4 and the virus host shut-off function. The design of a suitable promoter regulator capable of expressing foreign genes during latency must also be developed. Many promoters including strong viral promoters and neuronal-specific cellular promoters have proved to be inadequate due to the transient nature of their activity from the viral vector genome and the viral latency promoter region appears to be weakly active in brain. Current efforts concern the development of auto-regulatable promoters which can remain active even if the viral genome is bound by chromatin.

Base Sequence↗
PubMed1993

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C.

Entry of herpes simplex virus (HSV) into cells is believed to be mediated by specific binding of envelope proteins to a cellular receptor. Neomycin specifically blocks this initial step in infection by HSV-1 but not HSV-2. Resistance of HSV-2 to this compound maps to a region of the genome encoding glycoprotein C (gC-2). We have studied the function of gC-2 in the initial interaction of the virus with the host cell, using HSV-2 mutants deleted for gC-2 and gC-2-rescued recombinants. Resistance to neomycin was directly linked to the presence of gC-2 within the viral genome. In addition, deletion of the gC-2 gene caused a marked delay in adsorption to cells relative to the wild-type virus. HSV-1 recombinants containing chimeric gC genes composed of HSV-1 and HSV-2 sequences were used to localize neomycin resistance within the N-terminal 223 amino acids of gC-2. This region of the glycoprotein comprises an important domain responsible for binding of HSV-2 to cell receptors in the presence of neomycin. A gC-2-negative mutant is still infectious, indicating that HSV-2 also has an alternative pathway of adsorption.

Animals↗
PubMed1992

In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer.

Stereotactic inoculation of a herpes simplex virus (HSV) gene transfer vector into the hippocampus and caudate of rat brain resulted in limited and transient viral replication and the establishment of latency. Virus attenuation was achieved by insertional inactivation of a viral gene, Us3. Insertion of a lacZ reporter gene, under the control of the HSV glycoprotein C (gC) late gene promoter, allowed viral replication to be monitored in vivo. Unlike unattenuated virus, the Us3::pgC-lacZ recombinant caused little apparent damage to normal hippocampal morphology. Transient lacZ expression was detected in a considerable population of neurons of the dentate gyrus following hippocampal injection, whereas few positively staining neurons were present within the caudate after injection at that site. Latency-associated transcripts, the hallmark of latent infection, were detected in the brain 10 months after injection. This recombinant virus may be useful as a gene transfer vector for long-term expression of foreign genes in the central nervous system.

Animals↗
PubMed1992

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.

Amino Acid Sequence↗
PubMed1991

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Amino Acid Sequence↗
PubMed1986

Expression of a human cytomegalovirus late gene is posttranscriptionally regulated by a 3'-end-processing event occurring exclusively late after infection.

A phenomenon of posttranscriptional regulation has been previously identified in cytomegalovirus-infected human fibroblast cells (Wathen and Stinski, J. Virol. 41:462, 1982). A region typifying this phenomenon has been located within the large unique component of the viral genome (map units 0.408 to 0.423). Even though this transcriptional unit was highly transcribed at early times after infection, mRNAs from this region were only detectable on the polyribosomes after viral DNA replication. Thus, this region is believed to code for a late gene. Single-strand-specific nuclease mapping experiments of viral transcripts established that the transcriptional initiation sites and the 5' ends of a downstream exon were identical at early and late times. However, the late transcripts differed from the early transcripts by the processing of the 3' end of the viral RNAs. This involved either the removal of a distinct region of the transcript by the selection of an upstream cleavage and polyadenylation site or the differential splicing of the RNA molecule. The upstream cleavage and polyadenylation site was identified by nuclease mapping analyses and DNA sequencing. The 3'-end processing of these transcripts is necessary for the detection of these viral RNAs within the cytoplasm of the infected cell. We propose that human cytomegalovirus either codes for a factor(s) that is involved in the 3'-end-processing event at late times after infection or stimulates the synthesis of a host cell factor(s) involved in this complex regulatory event. This level of regulation may have an influence on the types of cells that permit productive cytomegalovirus replication.

Base Sequence↗
PubMed1984

Promoter-regulatory region of the major immediate early gene of human cytomegalovirus.

The DNA templates containing immediate early (IE) genes of human cytomegalovirus (CMV) were transcribed in vitro by using a HeLa cell extract. When IE region 1, 2, and 3 were used, transcription was detected qualitatively only from IE region 1. Transcription was detected with DNA representing IE region 2 when the IE region 1 promoter was not present. DNA sequence analysis of the upstream regulatory region of IE region 1 detected two distinct repeats of 19 and 18 nucleotides, both being repeated four times. A putative cruciform structure could form through the surrounding sequences with each 18-nucleotide repeat being located in the unpaired region. The potential secondary structure and the repeat sequences in the regulatory region of IE region 1 are presumably related to the high level of transcription of this IE gene.

Base Sequence↗
PubMedDate not supplied

Herpes simplex virus vector-mediated dystrophin gene transfer and expression in MDX mouse skeletal muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) results from mutations that prevent the expression of functional dystrophin in muscle fibers. Herpes simplex virus type-1 (HSV-1) represents a potentially useful vector for treatment of DMD because it has the capacity to accommodate the 14-kb full-length dystrophin cDNA and can efficiently transduce muscle cells. We have tested the ability of first- and second-generation replication-defective HSV vectors to deliver full-length dystrophin to dystrophin-deficient mdx muscle cells in vitro and in vivo. METHODS: First-generation replication-defective HSV vectors harboring full-length or truncated (Becker) dystrophin expression cassettes and lacking a single viral immediate-early (IE) gene were constructed and tested by immunofluorescence and immunoblotting for their ability to direct dystrophin expression in infected mdx cells in culture. To reduce vector cytotoxicity and safety concerns, a second-generation dystrophin vector missing additional IE genes was constructed and tested in vitro and in vivo. RESULTS: Dystrophin expression was observed in infected mdx myotubes in vitro in all cases. Confocal microscopy showed exclusive localization of full-length dystrophin to the cell membrane whereas the Becker variant was also found abundantly throughout the cytoplasm. Dystrophin expression in mdx mice was restored in muscle cells near the site of vector injection. CONCLUSION: Highly defective HSV-1 vectors which lack the ability to spread systemically and are greatly reduced in toxicity for infected cells, thus removing an impediment to prolonged transgene expression, can direct the delivery and proper expression of full-length dystrophin whose considerable size is compatible with few other modes of delivery. These vectors may offer a legitimate opportunity toward the development of effective gene therapy treatments for DMD.

Animals↗