Biomedical subjects

Kevin V Morris

Publications and source records attributed to Kevin V Morris.

15 recordsLinked to original sources

PubMed2006

Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells.

Argonaute proteins are the core components of effector complexes that facilitate RNA interference (RNAi). Small interfering RNAs (siRNAs) targeted to promoter regions mediate transcriptional gene silencing (TGS) in human cells through heterochromatin formation. RNAi effector complexes have yet to be implicated in the mechanism of mammalian TGS. Here we describe the role of the human Argonaute-1 homolog (AGO1) in directing TGS at the promoters for human immunodeficiency virus-1 coreceptor CCR5 and tumor suppressor RASSF1A. AGO1 associates with RNA polymerase II (RNAPII) and is required for histone H3 Lys9 dimethylation and TGS. AGO1, TAR RNA-binding protein-2 (7TRBP2) and Polycomb protein EZH2 colocalize to the siRNA-targeted RASSF1A promoter, implicating Polycomb silencing in the mechanism of mammalian TGS. These results establish a connection between RNAi components AGO1 and TRBP2, RNAPII transcription and Polycomb-regulated control of gene expression.

Argonaute Proteins↗

PubMed2006

Are viral-encoded microRNAs mediating latent HIV-1 infection?

The Human Immunodeficiency Virus type 1 (HIV-1), a member of the lentivirus subfamily, infects both dividing and nondividing cells and, following reverse transcription of the viral RNA genome, integrates into the host chromatin where it enters into a latent state. Many of the factors governing viral latency remain unresolved and current antiviral treatment regimens are largely ineffective at eliminating cellular reservoirs of latent virus. The recent identification of microRNA (miRNA) encoding sequences embedded in the HIV-1 genome, and the discovery of functional virus-derived miRNAs, suggests a role for RNA Interference (RNAi) in the regulation of HIV-1 gene expression. Recently, the mammalian RNAi machinery was shown to regulate gene expression epigenetically by transcriptional modulation, providing a direct link between RNAi and a mechanism for inducing latency. Interestingly, both HIV-1 Tat, and the host TAR RNA-binding protein (TRBP), bind to the transactivating response (TAR) RNA of HIV-1 and affect the function of RNAi in human cells. Specifically, TRBP, a cofactor in Tat-TAR interactions, is a vital component of Dicer-mediated dsRNA processing. These novel observations support a central role for HIV-1 and associated host factors in regulating cellular RNAi and viral gene expression through RNA directed processes. Thus, HIV-1 may have evolved mechanisms to exploit the RNAi pathway at both the transcriptional and posttranscriptional level to affect and/or maintain a latent infection.

Gene Expression Regulation, Viral↗

PubMed2006

Lentivirus-mediated RNA interference therapy for human immunodeficiency virus type 1 infection.

RNA interference (RNAi) is a natural mechanism by which small interfering RNAs (siRNAs) operate to specifically and potently downregulate the expression of a target gene. This downregulation has been demonstrated by targeting siRNAs to the mRNA (posttranscriptional gene silencing) as well as to the gene promoter, regulating gene expression epigenetically by transcriptional gene silencing. These observations significantly broaden the role RNA plays in the cell and suggest that siRNAs could prove to be a potent future therapeutic for the treatment of diseases such as human immunodeficiency virus type 1 (HIV-1) infection. The specificity and simplicity of design and the ability to express siRNAs from mammalian promoters make the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a soon-to-be-realized technology. However, the delivery and stable expression of siRNAs to target cells remain an enigma that could be surmounted, at least regarding the treatment of HIV-1 infection, by the application of lentiviral vectors to deliver and express anti-HIV-1 siRNAs in target cells. This review focuses on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating HIV-1.

Genetic Therapy↗

PubMed2006

Therapeutic potential of siRNA-mediated transcriptional gene silencing.

RNA interference (RNAi) and specifically the use of small interfering RNAs (siRNAs) represents a potentially new paradigm in gene knockout technology. Clearly siRNAs can be used to knockdown the expression of a targeted transcript in what has been termed posttranscriptional gene silencing (PTGS). While there are a plethora of reports applying siRNA-mediated PTGS the limitation of the duration of the effect remains. Recently, in human cells, siRNAs have been shown, similar to plants and Schizosaccharomyces pombe, to mediate transcriptional gene silencing (TGS). The observation that siRNAs can function in a TGS manner in human cells suggests that, similar to plants, human genes may also be able to be silenced more permanently via epigenetic modifications. The ramifications of siRNA-mediated TGS in humans suggest that longer term suppression of gene function can be obtained via siRNA-directed chromatin modifications. Undoubtedly the potential to employ siRNA technology is broader than once envisioned in human cells and suggests that siRNA-mediated TGS is not simply limited to PTGS. The potential to utilize siRNAs to direct epigenetic changes in local chromatin structure offers a new therapeutic avenue that could prove remarkably robust and of immeasurable therapeutic value in the directed control of target gene expression.

Animals↗

PubMed2006

Antiviral applications of RNAi.

RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) operates to specifically and potently downregulate the expression of a target gene. This downregulation has been thought to predominantly function at the level of mRNA, as post-transcriptional gene silencing. The discovery that siRNAs can suppress gene expression at the level of transcription, that is, transcriptional gene silencing, has created a major paradigm shift in mammalian RNAi. These findings significantly broaden the role that RNA, specifically siRNA and potentially microRNA, plays in the regulation of gene expression, as well as the breadth of potential siRNA target sites. Indeed, the specificity and simplicity of design makes the use of siRNAs to target and suppress virtually any gene of interest a realized technology. Furthermore, since siRNAs are small nucleic acid reagents, they are unlikely to elicit an immune response, theoretically making them good therapeutics. The development, delivery and potential therapeutic use of antiviral siRNAs in treating viral infections and emerging viral threats are reviewed.

Animals↗

PubMed2005

The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells.

To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to alpha-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.

Amanitins↗

PubMed2005

siRNA induced transcriptional gene silencing in mammalian cells.

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. It has recently become clear in human cells that RNA plays a far more profound and complex role in regulating the expression of the gene. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by small-interfering RNAs (siRNAs). Specifically, the recent observations by both our groups that siRNAs can silence target genes at the level of the chromatin in mammalian cells. We discuss here siRNA mediated transcriptional gene silencing and directed DNA methylation as well as the putative mechanism involved in human cells. Undoubtedly, the ramifications from this paradigm shift of RNA regulating the expression of the gene are immeasurable both therapeutically (i.e., directed control of a genes expression) and biologically in understanding the evolution of the cell.

Animals↗

PubMed2005

Inhibition of HIV-1 replication by siRNA targeting conserved regions of gag/pol.

Short interfering RNAs (siRNAs) targeting HIV-1gag, vif, tat, rev and host CD4 and CCR5 have been reported to inhibit HIV replication. However, the sequence divergence of HIV and the concentration dependence of siRNA activity represent significant challenges to RNAi mediated inhibition. To determine the parameters of RNAi in suppression of HIV-1 we screened seven siRNA candidates targeting highly conserved regions of gag/pol, based on target site GC content, for antiviral activity at varying concentrations. Only two of these inhibited CA-p24 production more than 50%, 2064 and 2161. Activity varied with concentration, with 100 nM producing optimal suppression. Requirements for target sequence conservation and activity over a range of concentrations may severely limit the number of siRNA candidates for therapeutic development.

Cell Line↗

PubMed2005

Characterization of human immunodeficiency virus (HIV)-2 vector mobilization by HIV-1.

Conditionally replicating human immunodeficiency virus type 2 (crHIV-2) vectors can compete with HIV-1 for packaging in HIV-1-infected cells, indicating that the mobilization of vectors could selectively target as well as protect reservoirs susceptible to HIV-1 infection. The incorporation of HIV-1-specific antiviral transgenes in crHIV-2 vectors, although increasing the direct antiviral effect, may decrease mobilization and transmission to surrounding cells. To investigate how HIV-1-specific catalytic RNA cassettes (ribozymes) affect this balance between antiviral activity and mobilization, crHIV-2 vectors shown to display anti-HIV-1 activity were packaged by HIV-2 and used to transduce cells previously infected with HIV-1 or to transduce uninfected cells that were subsequently challenged with HIV-1. Vector mobilization was greater when HIV-1-infected cells were transduced with vector than when transduced cells were infected with HIV-1, and approximately 3-fold lower vector production was observed in cultures transduced with vectors expressing anti-HIV-1 ribozymes. Vector and antiviral effects could be transferred to new cultures by passaging supernatants to fresh cultures. No evidence of recombination with HIV-1 was observed. Vector mobilization and protection from HIV-1 infection were also demonstrated in human peripheral blood mononuclear cells. These data suggest that strategies employing vector mobilization for HIV-1 gene therapy should use vectors with maximal antiviral potency, despite resulting reductions in mobilization of the vector.

Base Sequence↗

PubMed2005

VRX-496(VIRxSYS).

VIRxSYS is developing VRX-496, a lentiviral HIV-based vector encoding anti-HIV antisense envelope sequences, as a potential gene therapy for HIV infection. In July 2003, VIRxSYS undertook the initial dosing of an HIV-positive patient in a phase I/IIa trial.

AIDS Vaccines↗

PubMed2004

Small interfering RNA-induced transcriptional gene silencing in human cells.

Small interfering RNA (siRNA) and microRNA silence genes at the transcriptional, posttranscriptional, and/or translational level. Using human tissue culture cells, we show that promoter-directed siRNA inhibits transcription of an integrated, proviral, elongation factor 1alpha (EF1A) promoter-green fluorescent protein reporter gene and of endogenous EF1A. Silencing was associated with DNA methylation of the targeted sequence, and it required either active transport of siRNA into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction. These results demonstrate that siRNA-directed transcriptional silencing is conserved in mammals, providing a means to inhibit mammalian gene function.

Active Transport, Cell Nucleus↗

PubMed2004

Transduction of cell lines and primary cells by FIV-packaged HIV vectors.

Human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV) are capable of packaging viral RNA derived from heterologous as well as homologous lentiviruses, a phenomenon referred to as "cross packaging." To remove the possibility of seroconversion to HIV proteins, and to avoid potential problems arising due to targeting of vector or packaging construct by antiviral genes, we investigated the feasibility of using an FIV-based packaging system to deliver human immunodeficiency virus type 2 (HIV-2)-based vectors bearing anti-HIV-1 RNA expression cassettes to target cells. In the absence of FIV rev, FIV was packaged by HIV-2 at only 3% the efficiency of FIV packaging by FIV, but this was increased to 39% of homologous controls by supplying FIV rev in trans. HIV-2 vectors were packaged by FIV at levels equal to or exceeding the homologous HIV-2 packaging system in the absence of HIV-1 tat and rev, and levels increased approximately four- to fivefold with the addition of tat and rev in trans. HIV-2 vectors bearing a polyribozyme cassette targeting multiple regions of HIV RNA were efficiently packaged by FIV and transferred to target cells. Upon challenge with cell-free HIV-1 (m.o.i. = 0.1) a significant reduction in replication was observed. These findings demonstrate that packaging HIV vectors with FIV is a viable alternative, which avoids use of HIV structural proteins.

Cell Line↗

PubMed2004

Characterization of a mobilization-competent simian immunodeficiency virus (SIV) vector containing a ribozyme against SIV polymerase.

Exploitation of the intracellular virus machinery within infected cells to drive an anti-viral gene therapy vector may prove to be a feasible alternative to reducing viral loads or overall virus infectivity while propagating the spread of a therapeutic vector. Using a simian immunodeficiency virus (SIV)-based system, it was shown that the pre-existing retroviral biological machinery within SIV-infected cells can drive the expression of an anti-SIV pol ribozyme and mobilize the vector to transduce neighbouring cells. The anti-SIV pol ribozyme vector was derived from the SIV backbone and contained the 5'- and 3'LTR including transactivation-response, Psi and Rev-responsive elements, thus requiring Tat and Rev and therefore limiting expression to SIV-infected cells. The data presented here show an early reduction in SIV p27 levels in the presence of the anti-SIV pol ribozyme, as well as successful mobilization (vector RNA constituted approximately 17 % of the total virus pool) and spread of the vector containing this ribozyme. These findings provide direct evidence that mobilization of an anti-retroviral SIV gene therapy vector is feasible in the SIV/macaque model.

Base Sequence↗

PubMed2004

Anti-HIV-1 gene expressing lentiviral vectors as an adjunctive therapy for HIV-1 infection.

Lentiviral based gene therapy may provide a valuable addition to the current anti-HIV arsenal. Many lentiviral vector systems have been described including those based on feline immunodeficiency virus (FIV), human immunodeficiency virus 1 (HIV) and 2 (HIV-2/SIV) as well as replication incompetent, self-inactivating (sin) vs. conditionally replicating (mobilizable) vectors. Lentiviral vectors offer promise in treating HIV-1 infection as they are capable of stably transducing both dividing and nondividing cells, specifically those cells involved in HIV-1 replication and immune restoration: T-cells, hematopoietic stem cells, and dendritic cells. Moreover, some of the HIV-1 and 2 based vectors can be mobilized by wildtype HIV-1 in vivo and spread to those cells targeted by the virus as well as can compete with viral RNA for packaging and access to viral proteins such as Tat and Rev required for viral replication. Finally, lentiviral vectors can be designed to express therapeutic anti-HIV-1 genes, which specifically target various stages of viral replication. Many candidate RNA based anti-HIV-1 genes have been expressed from lentiviral vectors including ribozymes and anti-sense RNA [1]. Recently, small interfering RNAs (siRNAs) have been shown to potently suppress HIV replication [2-6]. This review will focus on the current status of lentiviral vector development and the feasibility of using lentiviral vectors in delivering anti-HIV genes, specifically ribozymes, and siRNAs as a therapeutic approach to employ in conjunction with current anti-retroviral therapies.

Base Sequence↗

PubMed2003

The effects of HHV-8 vMIP-II on SIVmac251 infection and replication competent and incompetent SIVmac239Delta3 vectors.

Human herpesvirus type 8 vMIP-II has one of the broadest ranges of chemokine receptor binding and therefore a multiplicity of biologic effects, both immunologic and antiviral. These properties make vMIP-II an attractive effector gene to be expressed from gene therapy vectors. The present studies were concerned with both therapeutic approaches: (1) an anti-simian immunodeficiency virus (SIV) biologic, and (2) an effector gene in SIV-based vectors. Regarding its antiviral properties, vMIP-II expressed from bacteria and SIV-based vectors bound the surface of CEMx174 cells and specifically suppressed SIVmac251 infection. A CCR3 monoclonal antibody partially inhibited vMIP-II binding, suggesting that both SIVmac251 and vMIP-II utilize a similar CCR3-like receptor for CEMx174 cell binding. Replication competent SIV-based vectors containing forward and reverse vMIP-II produced neither identifiable vMIP-II nor virions for the first 21 days. Virus replication occurred after this period. Significant sequence alterations in the forward vMIP-II containing replication competent vector transcripts were responsible for the failure of vMIP-II expression. The genetic basis for the initial failure to replicate virus and its later restoration was not determined but appeared in the II-PIMv containing vectors to coincide with deletions and compensatory rearrangements in nef 3' of the polypurine tract. Cells transfected with SIVmac239Delta3DeltaLTR-vMIP-II vectors expressed biologically active vMIP-II that bound CEMx174 cells and suppressed SIVmac251 infection. These data suggest that replication defective SIV vectors expressing immunobiolgic genes such as vMIP-II may prove useful in gene therapies, particularly in augmenting immune responses in chronically infected individuals.

Anti-HIV Agents↗