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Ali Ramezani

Publications and source records attributed to Ali Ramezani.

14 recordsLinked to original sources
PubMed2006

Assessment of an anti-HIV-1 combination gene therapy strategy using the antisense RNA and multimeric hammerhead ribozymes.

A combination gene therapy strategy using an ASPsi-gag antisense RNA (targeted against the packaging signal and the gag-coding region) and a multimeric hammerhead ribozyme Rz1-9 (targeted against nine sites within the env-coding region) or Rz1-14 (targeted against 14 sites within the 5' leader and the pro-, pol-, vif- and env-coding regions) was assessed for inhibiting HIV-1 replication. A murine stem cell virus (MSCV)-based MGIN vector was used to express Rz1-9, Rz1-14, ASPsi-gag, Rz1-9ASPsi-gag, or Rz1-14ASPsi-gag RNA in a CD4+ T lymphoid cell line. Stable transductants were shown to express similar levels of interfering RNA. HIV-1 replication was inhibited in cells expressing Rz1-9 and Rz1-14. Little inhibition of HIV-1 replication was observed in cells expressing ASPsi-gag RNA. Thus, the multimeric hammerhead ribozymes inhibit HIV-1 replication better than the antisense RNA. Inhibition of HIV-1 replication in cells expressing Rz1-9ASPsi-gag or Rz1-14ASPsi-gag RNA was worse than that obtained with the multimeric ribozymes alone. This result suggests that co-expression of antisense RNA decreases the anti-HIV potential of ribozymes. The multimeric ribozymes and the antisense RNA were designed to target different sites within the HIV-1 RNA. They are not expected to interact with each other. Neither are they expected to compete with each other for binding to the HIV-1 RNA. Instead, the antisense RNA binding to its (1553 nt-long) target site may have resulted in a decreased ribozyme turn over. Furthermore, since the antisense RNA/HIV-1 RNA hybrids are degraded by the cells, the co-expressed antisense RNA may have led to ribozyme degradation.

CD4-Positive T-Lymphocytes↗
PubMed2006

Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development.

Unlike conventional gammaretroviral vectors, the murine stem cell virus (MSCV) can efficiently express transgenes in undifferentiated embryonic stem cells (ESCs). However, a dramatic extinction of expression is observed when ESCs are subjected to in vitro hematopoietic differentiation. Here we report the construction of a self-inactivating vector from MSCV, MSinSB, which transmits an intron embedded within the internal transgene cassette to transduced cells. The internal transgene transcriptional unit in MSinSB comprises the composite cytomegalovirus immediate early enhancer-chicken beta-actin promoter and associated 5' splice site positioned upstream of the natural 3' splice site of the gammaretroviral envelope gene, and is configured such that the transgene translational initiation sequence is coincident with the envelope ATG. MSinSB could be produced at titers approaching 10(6) transducing units/ml and directed higher levels of transgene expression in ESCs than a splicing-optimized MSCV-derived vector, MSGV1. Moreover, when transduced ESCs were differentiated into hematopoietic cells in vitro, MSinSB remained transcriptionally active in greater than 90% of the cells, whereas MSGV1 expression was almost completely shut off. Persistent high-level expression of the MSinSB gammaretroviral vector was also demonstrated in murine bone marrow transplant recipients and following in vitro myelomonocytic differentiation of human CD34(+) cord blood stem/progenitor cells.

Animals↗
PubMed2006

Hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols, the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines.

Animals↗
PubMed2005

Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells.

We attempted to extend the lifespan of CD34+ stem/progenitor cells in human cord blood (CB) by transduction with lentiviral vectors carrying the human telomerase catalytic subunit (hTERT) and/or the human papillomavirus type 16 (HPV16) E6 and E7 oncogenes. We found that hTERT was incapable of prolonging the replicative capacity of CB cells maintained under serum-free conditions in the presence of stem cell factor, Flt3 ligand, thrombopoietin, and interleukin-3 beyond 4 months (n=3). However, transduced CB cells cultured in the same cytokine cocktail constitutively expressing HPV16 E6/E7 alone (n=2) or in concert with hTERT (n=9) continued to proliferate, giving rise to permanent (>2 years) cell lines with a CD45+ CD34- CD133+/- CD44+ CD235a+ CD71+ CD203+ CD33+ CD13+ myeloerythroid/mast cell progenitor phenotype. Notably, CB cell cultures expressing only HPV16 E6/E7 went through a crisis period, and the resulting oligoclonal cell lines were highly aneuploid. By comparison, the CB cell lines obtained by coexpression of HPV16 E6/E7 plus hTERT exhibited near-diploid karyotypes with minimal chromosomal aberrations, concomitant with stabilization of telomere length, yet were clonally derived. The immortalized E6/E7 plus hTERT-expressing CB cells were not tumorigenic when injected intravenously or subcutaneously into sublethally irradiated immunodeficient nonobese diabetic/severe combined immunodeficient mice but could be converted to a malignant state by ectopic expression of a v-H-ras or BCR-ABL oncogene. These findings provide new insights into the mechanisms governing the senescence checkpoint of primitive human hematopoietic precursors and establish a paradigm for studies of the multistep process of human leukemogenesis.

Antigens, CD34↗
PubMed2005

Specific pharmacological dimerization of KDR in lentivirally transduced human hematopoietic cells activates anti-apoptotic and proliferative mechanisms.

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context, little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional, biochemical, and microarray analyses. When deprived of natural ligands, GM-CSF, interleukin-3, or erythropoietin, AP20187 prevented apoptosis of transduced TF1 cells, induced dose-dependent proliferation, and supported long-term growth. In addition, AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably, mRNAs affiliated with heat stress, including Hsp70 and Hsp105, were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence, in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor, or KDR, signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes.

Apoptosis↗
PubMed2004

Tissue inhibitor of matrix metalloproteinase-1 overexpression in M1 myeloblasts impairs IL-6-induced differentiation.

The balance between matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) is important for extracellular matrix interactions of hematopoietic cells. MMP-independent growth modulating activity for TIMP-1 on B lymphocytes and erythroid progenitors has also been described, but a role for TIMP-1 in myelomonocytic differentiation has not been previously reported. In this study, we demonstrate that TIMP-1 overexpression impairs differentiation of the myeloblastic M1 cell line following interleukin (IL)-6 stimulation. We generated retroviral vectors coexpressing human TIMP-1 and the green fluorescent protein (GFP) and stably transduced murine M1 myeloid cells. TIMP-1 expressing cells showed a large reduction in IL-6-induced macrophage differentiation in vitro that was reversible with a specific monoclonal antibody. The differentiation delay in M1/TIMP-1 cells was also specifically reversible by pharmacologic phosphatidylinositol-3 kinase (PI3-K) inhibition. Additionally, overexpression of a TIMP-1/GFP fusion protein also impaired M1 differentiation and this protein was localized to the cell surface, consistent with an autocrine receptor-mediated mechanism. Surprisingly, TIMP-1 transduced cells had a selective advantage for growth in IL-6, indicating that functional effects on growth and differentiation of M1 cells were primarily through an autocrine mechanism. Intrinsic TIMP-1 expression in myeloid leukemia cells might thus impact upon survival or differentiation.

Bone Marrow Cells↗
PubMed2004

Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.

Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.

Animals↗
PubMed2004

Detection and enrichment of hematopoietic stem cells by side population phenotype.

A flow cytometric procedure has recently been described to isolate hematopoietic stem cells from mouse bone marrow based on the efflux properties of the vital dye Hoechst 33342. The assay defines a subset of cells-termed the "side population" (SP)-by simultaneously measuring fluorescence of the dye at two wavelengths (~450 nm and >670 nm). In this chapter, SP protocols are provided to detect candidate hematopoietic stem cells in mouse bone marrow and human cord blood. In the standard method, SP profiles are readily observed on a stream-in-air cell sorter using 30 mW of 351-356 nm ultraviolet excitation from a krypton-ion laser. Alternatively, SP profiles can be resolved on an analytical flow cytometer with cuvette flow cell using 8 mW of 325-nm ultraviolet excitation from a helium-cadmium laser. The ability to perform the SP assay on an analytical instrument facilitates optimization of staining conditions to identify hematopoietic and other stem cells in a variety of tissues. It is also demonstrated that SP profiles of slightly lower resolution can be obtained on a stream-in-air cell sorter using 100 mW of 407-nm violet excitation from a krypton-ion laser, raising the possibility that with appropriate validation the SP assay could be performed on flow cytometers that are not equipped with ultraviolet lasers.

Animals↗
PubMed2003

Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator.

Retroviral vectors are the most efficient means of stable gene delivery to hematopoietic stem cells (HSCs). However, transgene expression from retroviral vectors is frequently subject to the negative influence of chromosomal sequences flanking the site of integration. Toward the development of autonomous transgene expression cassettes, we inserted the human interferon-beta scaffold attachment region (IFN-SAR) and the chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator both separately and together into a series of self-inactivating (SIN) lentiviral vector backbones. Transduced cells of the human CD34+ hematopoietic progenitor line KG1a-pooled populations as well as individual clones harboring single integrants--were analyzed for reporter expression during culture periods of up to 4 months. Vectors carrying both the 5'HS4 insulator and the IFN-SAR consistently outperformed control vectors without inserts as well as vectors carrying either element alone. The performance of a set of vectors containing the murine stem cell virus long terminal repeat as an internal promoter was subsequently assessed during in vitro monocytic differentiation of transduced primary human CD34+ cord blood cells. Similar to what was observed in the KG1a hematopoietic progenitor cell model, optimal reporter expression in primary monocytes was obtained with the vector bearing both regulatory elements. These findings indicate that the 5'HS4/IFN-SAR combination is particularly effective at maintaining open chromatin domains permissive for high-level transgene expression at early and late stages of hematopoietic development, and thus could be of utility in HSC-directed retroviral vector-mediated gene transfer applications.

Animals↗
PubMed2003

Phenotype correction of Fanconi anemia group A hematopoietic stem cells using lentiviral vector.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure due to defective stem cell function. FA patients' cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC), exposure to which results in cytogenetic aberrations and cell death. To date Moloney murine leukemia virus vectors have been used in clinical gene therapy. Recently, third-generation lentiviral vectors based on the HIV-1 genome have been developed for efficient gene transfer to hematopoietic stem cells. We generated a self-inactivating lentiviral vector expressing the FA group A cDNA driven by the murine stem cell virus U3 LTR promoter and used the vector to transduce side-population (SP) cells isolated from bone marrow of Fanconi anemia group A (Fanca) knockout mice. One thousand transduced SP cells reconstituted the bone marrow of sublethally irradiated Fanca recipient mice. Phenotype correction was demonstrated by stable hematopoiesis following MMC challenge. Using real-time PCR, one proviral vector DNA copy per cell was detected in all lineage-committed cells in the peripheral blood of both primary and secondary recipients. Our results suggest that the lentiviral vector transduces stem cells capable of self-renewal and long-term hematopoiesis in vivo and is potentially useful for clinical gene therapy of FA hematopoietic cells.

Animals↗
PubMed2003

High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors.

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1alpha promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-beta gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.

Animals↗
PubMed2003

Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A.

Hemophilia A, the most common inherited bleeding disorder, is caused by deficiency or functional defects in coagulation factor VIII (fVIII). Conventional treatment for this disease involves intravenous infusions of plasma-derived or recombinant fVIII products. Although replacement therapy effectively stops the bleeding episodes, it has a risk of transmission of viral blood-borne diseases and development of neutralizing antibodies that inactivate the administered fVIII protein. Hemophilia A is an attractive candidate for application of gene therapy approaches because the therapeutic window is wide and even modest elevation of fVIII levels will correct the hemophilic phenotype. Ongoing preclinical investigations utilize animal models of hemophilia A, including genetically fVIII-deficient mice and naturally fVIII-deficient dogs, to optimize vectors, transgenes and target cell populations for Phase I clinical trials. In this review, we outline the progress in understanding the mechanisms of fVIII turnover, which provides a basis for development of improved fVIII molecules with prolonged half-life in the circulation. We discuss the possibility of incorporating these improved fVIII molecules as transgenes into self-inactivating lentiviral vectors carrying chromatin insulator sequences, representing a new generation of gene delivery vehicle, to target hematopoietic stem cells and endothelial cells. The use of hematopoietic stem cells as the target cell population may prevent inhibitor formation to transduced fVIII by induction of immune tolerance. Alternatively, endothelial cells may support optimal synthesis of fVIII and myeloablative conditioning of patients with radiation or chemotherapy may not be required for efficient engraftment of the engineered cells. Collectively, these proposed advances represent promising prophylactic strategies toward long-term correction of the coagulation defect in this progressively debilitating, life-threatening disease.

Animals↗
PubMed2002

Development and testing of retroviral vectors expressing multimeric hammerhead ribozymes targeted against all major clades of HIV-1.

Rz1-9 is a multimeric hammerhead ribozyme targeted against nine highly conserved sites within the env coding region of human immunodeficiency virus type-1 (HIV-1) RNA. This ribozyme was shown to inhibit HIV-1 replication (1, 2). However, Rz1-9 target sites are only conserved within the clade B of HIV-1. In this study, we have designed another multimeric ribozyme, Rz10-14. This multimeric ribozyme is targeted against five sites that are highly conserved amongst all major clades of HIV-1. A third multimeric ribozyme, Rz1-14, was obtained by combining both Rz1-9 and Rz10-14. A mouse stem cell virus-based MGIN vector (3) was used to express these ribozymes in a human CD4+ T lymphoid cell line. Stable transductants expressed vector RNA containing ribozymes which were shown to be active. In HIV-1 challenge experiments, very little or no virus production could be detected in the pools of stable MT4 transductants expressing Rz1-14 for 60 days tested. Inhibition of virus replication was most prominent with Rz1-14, followed by Rz1-9, and then Rz10-14. Thus, the combined multimeric ribozyme, Rz1-14, is more effective than Rz1-9 or Rz10-14. As Rz1-14 is targeted against all major clades of HIV-1, it will be further pursued for use in HIV-1 gene therapy.

CD4-Positive T-Lymphocytes↗