Publications by authors named "Lisa J Scherer"

6 Publications

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Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene.

Mol Ther 2014 May 28;22(5):952-63. Epub 2014 Feb 28.

1] Department of Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, California, USA [2] Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, California, USA.

Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMT(P140K) marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.
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http://dx.doi.org/10.1038/mt.2014.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015224PMC
May 2014

Ex vivo gene therapy for HIV-1 treatment.

Hum Mol Genet 2011 Apr 19;20(R1):R100-7. Epub 2011 Apr 19.

Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.

Until recently, progress in ex vivo gene therapy (GT) for human immunodeficiency virus-1 (HIV-1) treatment has been incremental. Long-term HIV-1 remission in a patient who received a heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5(-/-) donor, even after discontinuation of conventional therapy, has energized the field. We review the status of current approaches as well as future directions in the areas of therapeutic targets, combinatorial strategies, vector design, introduction of therapeutics into stem cells and enrichment/expansion of gene-modified cells. Finally, we discuss recent advances towards clinical application of HIV-1 GT.
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http://dx.doi.org/10.1093/hmg/ddr160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095057PMC
April 2011

Optimization and characterization of tRNA-shRNA expression constructs.

Nucleic Acids Res 2007 10;35(8):2620-8. Epub 2007 Apr 10.

Department of Molecular Biology Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte, California 91010, USA.

Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNA(Lys3)-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3' tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.
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http://dx.doi.org/10.1093/nar/gkm103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885648PMC
June 2007

Rapid assessment of anti-HIV siRNA efficacy using PCR-derived Pol III shRNA cassettes.

Mol Ther 2004 Sep;10(3):597-603

Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.

Identification of sequences within a target mRNA that are susceptible to potent siRNA knockdown often requires testing several independent siRNAs or shRNA expression cassettes. Using RNAi against HIV RNAs is further complicated by the length of the viral genome, the complexity of splicing patterns, and the propensity for genetic heterogeneity; consequently, it is most important to identify a number of siRNA targets that potently block viral replication. We previously described a facile PCR-based strategy for rapid synthesis of si/shRNA expression units and their testing in mammalian cells. Using this approach, which is rapid and inexpensive, it is possible to screen a number of potential RNAi targets in HIV to identify those that are most susceptible to RNAi. We report that shRNA expression cassettes constructed by PCR and cotransfected directly into mammalian cells with HIV proviral DNA express shRNAs that are inhibitory to HIV-1 replication. Our results also demonstrate that there is a wide range of efficacies among shRNAs targeting different sites throughout the HIV genome. By screening several different targets we were able to identify a sequence in a common tat/rev exon that is exquisitely sensitive to RNAi. Furthermore we relate the efficacies of our PCR product expressed shRNAs to the relative stabilities of the siRNA duplexes and the accessibilities of the target sites to antisense base pairing in cell extracts.
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http://dx.doi.org/10.1016/j.ymthe.2004.05.003DOI Listing
September 2004

Approaches for the sequence-specific knockdown of mRNA.

Nat Biotechnol 2003 Dec;21(12):1457-65

Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.

Over the past 25 years there have been thousands of published reports describing applications of antisense nucleic acid derivatives for targeted inhibition of gene function. The major classes of antisense agents currently used by investigators for sequence-specific mRNA knockdowns are antisense oligonucleotides (ODNs), ribozymes, DNAzymes and RNA interference (RNAi). Whatever the method, the problems for effective application are remarkably similar: efficient delivery, enhanced stability, minimization of off-target effects and identification of sensitive sites in the target RNAs. These challenges have been in existence from the first attempts to use antisense research tools, and need to be met before any antisense molecule can become widely accepted as a therapeutic agent.
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http://dx.doi.org/10.1038/nbt915DOI Listing
December 2003

Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.

Mol Ther 2002 Oct;6(4):481-9

Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.

We previously demonstrated that chimeric tRNA(Lys3)-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNA(Lys3) promoter variants and compared their expression levels from the internal tRNA(Lys3) promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNA(Lys3)-ribozyme containing a mutation in the tRNA B box. As detected by fluorescent in situ hybridization, transcripts from a construct with the tRNA promoter alone localized strictly to the cytoplasm, whereas transcripts from dual U6/tRNA promoter were present in both the cytoplasm and the nucleus. Inhibition of HIV-1 correlates well with expression levels of the chimeric constructs. The results presented demonstrate that U6 and tRNA promoters can be placed in tandem for high-level expression of small RNA therapeutic transcripts.
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http://dx.doi.org/10.1006/mthe.2002.0696DOI Listing
October 2002
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