High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results.

High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones.

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation.

Efficient targeted transduction of primary human endothelial cells with dual-targeted lentiviral vectors.

Angiogenesis is a rate-limiting factor for numerous human diseases. Angiogenic vessels and also the endothelium of certain organs such as the lung display molecular addresses that can be exploited for the selective delivery of gene therapeutics. Lentiviral vectors (LVs) are powerful tools for stable gene delivery but their integration and expression in undesired cell types poses a serious safety concern.

Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates.

RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34+ hematopoietic stem/progenitor cell transplant.

Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors.

Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. We examined the effects of shRNA expression in primary human lymphocytes (PBLs) using lentiviral vectors bearing different RNA polymerase III promoters. We found that the U6 promoter is more efficient than the H1 promoter for shRNA expression and for reducing expression of CCR5 in PBLs.

Induction of transgene-specific immunological tolerance in myeloablated nonhuman primates using lentivirally transduced CD34+ progenitor cells.

Modeling human hematopoietic progenitor cell gene therapy in nonhuman primates allows long-term evaluation of safety, maintenance of gene expression, and potential immune response against transgene products. We transplanted autologous G-CSF/SCF-mobilized CD34+ cells transduced with lentiviral vectors expressing EGFP into myeloablated rhesus macaques. To date, more than 4 years posttransplantation, 0.

Efficient lentiviral vectors for short hairpin RNA delivery into human cells.

RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription.