Biomedical subjects

R J Samulski

Publications and source records attributed to R J Samulski.

At least 73 records · Page 4Linked to original sources

PubMed1996

Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.

Muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and Duchene's muscular dystrophy. Previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. We introduced recombinant adeno-associated virus vector (rAAV) carrying a lacZ reporter into muscle tissue of immunocompetent mice. The lacZ reporter gene was efficiently transduced and expressed with no evidence of a cellular immune response. Moreover, gene expression persisted for more than 1.5 years. Molecular characterization of rAAV vector DNA suggests a mechanism for persistence, since vector episomes convert to high-molecular-weight genomic DNA. These data provide the first report for establishing long-term gene transduction into mammalian muscle cells in vivo without the need for immune modulation of the organism.

Animals↗

PubMed1996

Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors.

The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.

Adenoviridae↗

PubMed1995

AAV as a viral vector for human gene therapy. Generation of recombinant virus.

Investigation of the adeno-associated virus (AAV) life cycle has enabled the establishment of methodology and identification of critical cis-acting sequences required for recombinant AAV production. Vectors derived from the defective human parvovirus (AAV) have been used for successful gene transfer and expression in many diverse mammalian cell types, such as erythroid, airway epithelium, and neuronal cells. One of the crucial steps in the continued case of AAV as a vector is the development of packaging systems that will allow efficient encapsidation of foreign genes into AAV virions. For this reason, the focus of this article will be generation of recombinant AAV vectors.

Animals↗

PubMed1994

Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.

Antigens, CD↗

PubMed1994

Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells.

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta-galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral-associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.

Adenoviruses, Human↗

PubMed1994

Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.

Adeno-associated viral (AAV) vectors are non-pathogenic, integrating DNA vectors in which all viral genes are removed and helper virus is completely eliminated. To evaluate this system in the post-mitotic cells of the brain, we found that an AAV vector containing the lacZ gene (AAVlac) resulted in expression of beta-galactosidase up to three months post-injection in vivo. A second vector expressing human tyrosine hydroxylase (AAVth) was injected into the denervated striatum of unilateral 6-hydroxydopamine-lesioned rats. Tyrosine hydroxylase (TH) immunoreactivity was detectable in striatal neurons and glia for up to four months and we also found significant behavioural recovery in lesioned rats treated with AAVth versus AAVlac controls. Safe and stable TH gene transfer into the denervated striatum may have potential for the genetic therapy of Parkinson's disease.

Adenoviruses, Human↗

PubMed1994

Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector.

Fanconi anemia (FA) is a recessive inherited disease characterized by defective DNA repair. FA cells are hypersensitive to DNA cross-linking agents that cause chromosomal instability and cell death. FA is manifested clinically by progressive pancytopenia, variable physical anomalies, and predisposition to malignancy. Four complementation groups have been identified, termed A, B, C, and D. The gene for the FA complementation group C, FACC, has been cloned. Expression of the FACC cDNA corrects the phenotypic defect of FA(C) cells, resulting in normalized cell growth in the presence of DNA cross-linking agents such as mitomycin C (MMC). Gene transfer of the FACC gene should provide a survival advantage to transduced hematopoietic cells, suggesting that FA might be an ideal candidate for gene therapy. We demonstrated efficient transduction, expression, and phenotypic correction in lymphoblastoid cell lines derived from FA (C) patients using a recombinant adeno-associated virus (rAAV) vector containing the FACC gene. Molecular characterization of the transduced FACC gene showed an intact unrearranged proviral genome with expression sufficient to normalize cell growth, cell cycle kinetics and chromosomal breakage in the presence of MMC. These observations were extended by testing rAAV transduction in hematopoietic progenitor cells. Peripheral blood CD34+ cells isolated from a FA (C) patient and transduced with rAAV/FACC virus yielded 5-10-fold more progenitor colonies than mock-infected cells, consistent with genetic "rescue" of corrected cells. This is the first demonstration of rAAV gene correction in primary human hematopoietic progenitor cells and has important implications for gene therapy of hematopoietic disorders, specifically FA.

Base Sequence↗

PubMed1994

Expression of the human glucocerebrosidase and arylsulfatase A genes in murine and patient primary fibroblasts transduced by an adeno-associated virus vector.

We have constructed two recombinant adeno-associated virus (AAV) vectors (pJJ-3GC and pJJ-3ASA) which contained either the human glucocerebrosidase (GC) or arylsulfatase A (ASA) cDNA under the control of an SV40 promoter. These plasmids were co-transfected to 293 cells with a helper plasmid containing trans-acting AAV genes required for packaging the vectors. The two recombinant vectors successfully infected murine and patient fibroblasts. The human glucocerebrosidase and arylsulfatase A genes were expressed at high levels in the cells as determined by Western blot analysis, enzyme assay and immunochemical staining. GC enzyme activity in Gaucher patient fibroblasts (GM-0877) infected by AAV-GC was 15-fold higher than in non-infected cells. ASA enzyme activity in MLD 557g cells infected by AAV-ASA was up to 500-fold higher than in the metachromatic leukodystrophy (MLD) control cells. Southern blotting results showed that the vector integrated 1-2 copies of pJJ-3GC and ASA in the targeted cell genome. These two vectors will be useful in studying AAV-mediated transfer of the GC and ASA genes in cells and animals.

Animals↗

PubMed1993

Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer.

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut-NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single-copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

Binding Sites↗

PubMed1993

Adeno-associated virus: integration at a specific chromosomal locus.

Recent characterization of integration of the human parvovirus, adeno-associated virus, has uncovered the exciting occurrence of targeted integration. Thus far, such specific integration has been found to be unique among the eukaryotic viruses. The molecular details of the steps involved in virus integration are actively being pursued and should yield significant information for our understanding of the mechanisms of DNA transposition.

Animals↗

PubMed1993

Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein.

We previously demonstrated that the adeno-associated virus (AAV) Rep68 and Rep78 proteins are able to nick the AAV origin of DNA replication at the terminal resolution site (trs) in an ATP-dependent manner. Using four types of modified or mutant substrates, we now have investigated the substrate requirements of Rep68 in the trs endonuclease reaction. In the first kind of substrate, portions of the hairpinned AAV terminal repeat were deleted. Only deletions that retained virtually all of the small internal palindromes of the AAV terminal repeat were active in the endonuclease reaction. This result confirmed previous genetic and biochemical evidence that the secondary structure of the terminal repeat was an important feature for substrate recognition. In the second type of substrate, the trs was moved eight bases further away from the end of the genome. The mutant was nicked at a 50-fold-lower frequency relative to a wild-type origin, and the nick occurred at the correct trs sequence despite its new position. This finding indicated that the endonuclease reaction required a specific sequence at the trs in addition to the correct secondary structure. It also suggested that the minimum trs recognition sequence extended three bases from the cut site in the 3' direction. The third type of substrate harbored mismatched base pairs at the trs. The mismatch substrates contained a wild-type sequence on the strand normally cut but an incorrect sequence on the complementary strand. All of the mismatch mutants were capable of being nicked in the presence of ATP. However, there was substantial variation in the level of activity, suggesting that the sequence on the opposite strand may also be recognized during nicking. Analysis of the mismatch mutants also suggested that a single-stranded trs was a viable substrate for the enzyme. This interpretation was confirmed by analysis of the fourth type of substrate tested, which contained a single-stranded trs. This substrate was also cleaved efficiently by the enzyme provided that the correct strand was present in the substrate. In addition, the single-stranded substrate no longer required ATP as a cofactor for nicking. Finally, all of the substrates with mutant trss bound the Rep protein as efficiently as the wild-type did. This finding indicated that the sequence at the cut site was not involved in recognition of the terminal repeat for specific binding by the enzyme. We concluded that substrate recognition by the AAV Rep protein involves at least two and possibly as many as four features of the AAV terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1993

Gene therapy for human hemoglobinopathies.

Gene transfer of human globin genes into human pluripotent stem cells via viral vectors may soon be realized. The high level of globin gene expression believed to be required for the treatment of severe hemoglobinopathies necessitated the inclusion of cis-acting sequences (LCR). Retroviral vectors containing the LCR elements are prone to rearrangement, low titer, and poor expression. Inclusion of a "minilocus" containing four HS sites linked to a globin gene resulted in higher expression in transplanted mice, but rearrangement of the provirus still occurs, and it is unclear what significance these experiments have with regard to human marrow stem cell transduction. Recombinant AAV is among the newest of genetic transfer vectors. This once obscure virus possesses unique properties that distinguish it from all other vectors. Its major advantage is the lack of pathogenicity in humans. Wild-type AAV has the unusual ability to selectively integrate into the mammalian genome at a specific region, thus reducing the concern for genomic disruption and insertional mutagenesis. The ability of AAV to carry regulatory elements without interference from the viral template may enable greater control of transferred gene expression. Disadvantages currently include the inferior packaging systems which yield low numbers of recombinant virions which are contaminated with wild-type adenovirus. The small AAV genome that can be packaged (approximately 5 kb) rules out its use for transfer of larger genes. Recombinant AAV viruses do not appear to demonstrate the same site-specific genomic integration as wild-type viruses. Elucidation of the mechanism of site-specific integration should prove useful in the development of safe vectors for gene transfer as well as provide insight into the nature of DNA recombination in humans.

Dependovirus↗

PubMed1992

Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector.

Gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. Distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. We have explored the use of a human parvovirus, the adeno-associated virus (AAV), for globin gene transfer. The human A gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was subcloned into a plasmid (psub201) containing the AAV inverted terminal repeats. This construct was cotransfected with a helper plasmid containing trans-acting AAV genes into human 293 cells that had been infected with adenovirus. The recombinant AAV vector containing hypersensitivity site 2 stably introduced on average one or two unrearranged proviral copies into human K562 erythroleukemia cells. The transferred globin gene exhibited normal regulation upon hemin induction of erythroid maturation and was expressed at a level equivalent to a native chromosomal A gamma-globin gene.

Blotting, Southern↗

PubMed1992

Gene transfer in human lymphocytes using a vector based on adeno-associated virus.

Adeno-associated virus is a nonpathogenic, dependent parvovirus that integrates at a specific site in human chromosome 19. We have used the inverted terminal repeats of the virus, which mediate integration, to establish a vector for gene transfer in human lymphocytes. A neomycin resistance gene has been stably introduced into nontransformed human T-cell clones and a subsequent analysis of the functional properties of the infected clone revealed no detectable alterations. Rescue and replication of the wild-type virus was accomplished with adenovirus superinfection; however, the vector was not rescued and did not replicate by this procedure, indicating the stability of the integrated vector and demonstrating an additional level of safety incorporated in its construction. An adeno-associated virus-based vector represents an alternative to retroviruses for gene therapy in lymphocytes.

Base Sequence↗

PubMed1992

Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells.

The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically. In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells. By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins. In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci. Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells. These results suggest that replication centers similar to those established by other viruses exist for AAV. These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.

Antibodies, Monoclonal↗

PubMed1991

Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

A key feature in adeno-associated virus (AAV) replication is efficient integration of the viral genome into host cell DNA to establish latency when helper virus is absent. The steps involved in this process remain largely uncharacterized, even though AAV integration was first documented 20 years ago. Using a protein--DNA binding method we isolated AAV--cellular junction DNA sequences. The cellular component hybridized to a single restriction fragment in the virus-free parental cell line, and also co-migrated with AAV-specific sequences in numerous latently infected cell lines. Analysis of somatic cell hybrids indicated that this cellular sequence maps to the distal portion of the q arm of human chromosome 19. In situ hybridization of AAV DNA to chromosomes from latently infected cells confirms the physical location of AAV integrations to be q13.4-ter of chromosome 19. Sequence analysis of several independent integration sites shows breakpoints occurring within a 100 bp cellular region. This non-pathogenic parvovirus thus appears to establish viral latency by integrating its DNA specifically into one chromosomal region. Such specific integration is so far unique among the eukaryotic DNA viruses. The incorporation of site-specific integration into AAV vector schemes should make this vector system attractive for human gene therapy approaches.

Base Sequence↗

PubMed1990

In vitro resolution of covalently joined AAV chromosome ends.

We have developed an assay for a key step in the replication of adeno-associated virus (AAV) DNA. We demonstrate the covalently joined ends of linear AAV DNA can be resolved in vitro to the open duplex configuration. Only extracts prepared from human cells that have been infected with both adenovirus and AAV are capable of carrying out the reaction. The reaction is initiated by a site-specific and strand-specific endonucleolytic cut at a terminal resolution site near the end of the AAV terminal palindrome. During resolution the orientation of the terminal palindrome is inverted, and the 3' viral strand is extended by DNA synthesis. The size of the newly synthesized 3' strand is nearly identical to that found in viral particles. These observations provide direct biochemical evidence for an essential step in the model for AAV DNA replication.

Base Sequence↗

PubMed1990

Site-specific integration by adeno-associated virus.

Cellular sequences flanking integrated copies of the adeno-associated virus (AAV) genome were isolated from a latently infected clonal human cell line and used to probe genomic blots derived from an additional 21 independently derived clones of human cells latently infected with AAV. In genomic blots of uninfected human cell lines and of primary human tissue, each flanking-sequence probe hybridized to unique bands, but in 15 of the 22 latently infected clones the flanking sequences hybridized not only to the original fragments but also to a total of 36 additional species. AAV probes also hybridized to 22 of these new bands, representing 11 of the 15 positive clones, but never to the fragment characteristic of uninfected cell DNA. From these data we conclude that the AAV genome preferentially integrates into a specific region of the cellular genome. We have determined that the integration site is unique to chromosome 19 by somatic cell hybrid mapping, and this sequence has been isolated from uninfected human DNA.

Cell Line↗