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Biomedical subjects

Inder M Verma

Publications and source records attributed to Inder M Verma.

At least 19 recordsLinked to original sources
PubMed2003

BRCA2 cooperates with histone acetyltransferases in androgen receptor-mediated transcription.

Germ-line mutations of the BRCA2 tumor suppressor gene greatly increase the risk of developing breast and ovarian cancers. Here, we show that wild-type BRCA2, but not a tumor-specific truncated mutant BRCA2, synergizes with the nuclear receptor coactivator p160 GRIP1 to enhance transcriptional activation by androgen receptor (AR). BRCA2 not only associates with AR and GRIP1 but also cooperates with both the histone acetyltransferase P/CAF and BRCA1 to enhance AR- and GRIP1-mediated transactivation. As such, BRCA2 can exert its tumor suppressor function, in part, by modulating androgen signaling, which has been shown to be antiproliferative in a subset of breast cancer cells and particularly implicated in male breast tumors.

Acetyltransferases↗
PubMed2003

Neprilysin gene transfer reduces human amyloid pathology in transgenic mice.

The degenerative process of Alzheimer's disease is linked to a shift in the balance between amyloid-beta (Abeta) production, clearance, and degradation. Neprilysin has recently been implicated as a major extracellular Abeta degrading enzyme in the brain. However, there has been no direct demonstration that neprilysin antagonizes the deposition of amyloid-beta in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in transgenic mouse models of amyloidosis. We show that unilateral intracerebral injection of Lenti-Nep reduced amyloid-beta deposits by half relative to the untreated side. Furthermore, Lenti-Nep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice. These data further support a role for neprilysin in regulating cerebral amyloid deposition and suggest that gene transfer approaches might have potential for the development of alternative therapies for Alzheimer's disease.

Alzheimer Disease↗
PubMed2003

A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA.

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes in vitro and in vivo. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F(1) progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating "knockdown" mice will aid in functional genomics.

Animals↗
PubMed2003

Abrogation of postentry restriction of HIV-1-based lentiviral vector transduction in simian cells.

HIV-1 replication in simian cells is restricted at an early postentry step because of the presence of an inhibitory cellular factor. This block reduces the usefulness of HIV-1-based lentiviral vectors in primate animal models. Here, we demonstrate that substitution of the cyclophilin A (CyPA) binding region in the capsid of an HIV-1-based lentiviral vector (LV) with that of the macrophage tropic HIV-1 Ba-L resulted in a vector that was resistant to the inhibitory effect and efficiently transduced simian cells. Notably, the chimeric gag LV efficiently transduced primary simian hematopoietic progenitor cells, a critical cellular target in gene therapy. The alterations in the CyPA binding region did not affect CyPA incorporation; however, transduction by the gag chimeric LV seemed to be relatively insensitive to cyclosporin A, indicating that it does not require CyPA for early postentry steps. In dual infection experiments, the gag chimeric LV failed to remove the block to transduction of the WT LV, suggesting that the gag chimeric LV did not saturate the inhibitory simian cellular factor. These data suggest that the CyPA binding region of capsid contains a viral determinant involved in the postentry restriction of HIV-1-based lentiviral vectors. Overall, the findings demonstrate that the host range of HIV-1-based LV can be altered by modifications in the packaging construct.

Amino Acid Sequence↗
PubMed2003

Efficient production of human FVIII in hemophilic mice using lentiviral vectors.

Lentiviral vectors (LV) have the ability to integrate their proviral DNA containing a therapeutic gene into the host cell's genome. Therefore, these vectors have a great potential for gene therapy especially in the treatment of hereditary diseases like hemophilia A, which require lifelong expression of the transgene. We constructed an HIV-1-based LV containing human B-domain-deleted factor VIII (FVIII) cDNA under the control of a promoter consisting of the chicken beta-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which is a robust transcription promoter. High levels of FVIII expression from this vector could be demonstrated in vitro in 293T cells, primary liver cells, and hematopoietic progenitor cells. To test whether this viral vector was able to correct the bleeding disorder of C57BL/6 FVIII knockout mice, we transduced these mice with the FVIII LV either by intraperitoneal injection or by transplantation with transduced syngeneic bone marrow. FVIII production was analyzed in the blood plasma for a period of 3 months; however, only low levels of FVIII (<50 mU), which were below 5% of normal FVIII levels of 1000 mU, could be detected. Further analysis revealed that the low levels of FVIII activity present in the blood plasma were due to the presence of neutralizing antibodies to FVIII and not due to lack of expression of FVIII from the viral vector. FVIII expression could be detected in the tissues of the transduced mice by Western blot analysis and in ex vivo cultures. These data demonstrate that LVs are able to produce therapeutic levels of FVIII in knockout mice when administered by ip infection or by transduced hematopoietic cells. The challenge is to overcome the immune barriers to the therapeutic gene product.

Animals↗
PubMed2003

Good-bye Dolly.

Explore the source record for details and available documents.

Animals↗
PubMed2002

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein.

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.

BRCA1 Protein↗
PubMed2002

Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors.

Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.

Animals↗
PubMed2002

Restoration of spermatogenesis by lentiviral gene transfer: offspring from infertile mice.

Disruption of spermatogenesis found in azoospermia and oligozoospermia is thought to be of primarily genetic origin. Sl/Sl(d) mutant mice offer a model system in which lack of transmembrane type c-kit ligand (KL2) expression on the somatic Sertoli cell surface results in disruption of spermatogenesis. We investigated the ability of adeno-, adeno-associated-, retro-, and lentiviral vectors to transduce Sertoli cells and found that transduction with either adeno- or lentiviral vectors led to reporter gene expression for more than 2 mo after testicular tubule injection. Because adenoviral vectors showed toxicity, lentiviral vectors were used to express the c-kit ligand in Sl/Sl(d) Sertoli cells. Restoration of spermatogenesis was observed in all recipient testes. Furthermore, the sperm collected from recipient testes were able to generate normal pups after intracytoplasmic sperm injection. None of the offspring carried the transgene, suggesting the inability of lentiviral vectors to infect spermatogenic cells in vivo. We propose that lentiviral vectors can be used for gene therapy of male infertility without the risk of germ-line transmission.

Animals↗
PubMed2002

Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.

The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.

Animals↗
PubMed2002

Adeno-associated virus effectively mediates conditional gene modification in the brain.

The Cre/loxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates beta-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.

Animals↗
PubMed2002

Gene therapy with viral vectors.

A key factor in the success of gene therapy is the development of gene delivery systems that are capable of efficient gene transfer in a broad variety of tissues, without causing any pathogenic effect. Currently, viral vectors based on many different viruses have been developed, and their performance and pathogenicity has been evaluated in animal models. The results of these studies form the basis for the first clinical trials for correcting genetic disorders using retroviral, adenoviral, and adeno-associated viral vectors. Even though the results of these trials are encouraging, vector development is still required to improve and refine future treatment of hereditary disorders.

Gene Transfer Techniques↗