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. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5' and the 3' vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.

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. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.

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. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences) of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

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. We have developed a dual-targeted LV that can specifically target primary endothelial cells (ECs). Cell selectivity is achieved during entry, using a modified Sindbis virus envelope, and during transcription, with an EC-specific promoter. We evaluated four surface markers for EC targeting and seven promoter sequences from genes preferentially expressed in ECs. The efficiency and specificity of the double targeted vector were assayed in a panel of human primary cultures and tumor cell lines. A vector targeted to CD146, an endothelial adhesion molecule, and carrying a derivative of the EC tyrosine kinase Tie2 promoter, increased specificity of transduction up to 50 times and was also effective at selectively transducing ECs in a mixed coculture with human fibroblasts. The vector presented here is a potentially powerful tool that could be used in a variety of human diseases.

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. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1.

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. However, shRNA expression from the U6 promoter resulted in a gradual decline of the transduced cell populations. With one CCR5 shRNA this decline could be attributed to elevated apoptosis but another CCR5 shRNA that caused cytotoxicity did not show evidence of apoptosis, suggesting sequence-specific mechanisms for cytotoxicity. In contrast to the U6 promoter, PBLs transduced by vectors expressing shRNAs from the H1 promoter could be maintained without major cytotoxic effects. Since a lower level of shRNA expression appears to be advantageous to maintaining the shRNA-transduced population, lentiviral vectors bearing the H1 promoter are more suitable for stable transduction and expression of shRNA in primary human T lymphocytes. Our results suggest that functional shRNA screens should include tests for both potency and adverse metabolic effects upon primary cells.

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, 0.5-8% EGFP expression is maintained in multiple cell lineages. The animals remain healthy with no evidence of hematopoietic abnormalities or malignancies. To assess immune functions, we actively immunized two of our transplanted animals with purified rEGFP proteins and CpG adjuvant and demonstrated stable levels of EGFP+ cell populations maintained for over 29 months despite four active immunizations. We did not detect a persistent anti-EGFP antibody response or anti-EGFP T cell response in these immunized animals. Immune response to an irrelevant antigen was normal. Taken together, our data provide formal support that transplantation of lentivirally transduced CD34+ progenitor cells in myeloablated rhesus macaques induces specific immunological tolerance toward a foreign transgene.