Michaela Scherr

Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography.

Lentiviral vectors have been shown to stably transduce dividing and non-dividing target cells in vitro and in vivo. However, in vivo gene transfer applications with viral vectors in the central nervous system require highly efficient vector preparations, because only very small volumes can be injected stereotactically without damage to the brain tissue. Since lentiviral vectors are generated in transient co-transfection systems, viral preparations need to be purified and efficiently concentrated before injection into the brain. We describe an alternative procedure to concentrate lentiviral preparations by binding viral particles to an anion exchange column. Viral particles are eluted with sodium chloride, desalted and further concentrated by ultrafiltration. These vector preparations allowed high levels of gene transfer into terminally differentiated neuronal and glial cells and long-term transgene expression without any signs of acute and long-term toxicity or inflammation. The purification of lentiviral vectors from large-scale preparations by anion exchange chromatography allowed us to concentrate the virus to small volumes and to use these preparations to genetically modified target cells in vivo without signs of acute inflammatory responses.

Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo OLIGORIBONUCLEOTIDES AS THERAPEUTIC AGENTS

Two hammerhead ribozymes targeted to point mutations in codon 13 of the N-ras oncogene were synthesized and their catalytic activity and substrate specificity evaluated in vitro and ex vivo. In vitro studies showed that these ribozymes were specific for the oncogenic form of N-ras, since cleavage was observed only in a 849-nucleotide-long transcript containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2′-modified to protect them against degradation by nucleases. 2′-Fluoro-2′-deoxyuridine/cytidine-substituted ribozymes were nearly as active as their unmodified counterparts, but had a prolonged stability in cell culture supernatant containing fetal calf serum. The stability of the modified ribozymes increased by introduction of terminal phosphorothioates groups without significant influence in their catalytic efficiency. A sensitive assay based on the use of N-ras/luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nearly 60% in luciferase activity was observed in cells expressing mutant but not wild-type N-ras/luciferase fusion transcripts. Moreover, cleavage of N-ras transcripts in HeLa cells was directly confirmed by a semi-quantitative RT-PCR assay.

Retrovirally expressed anti-HIV ribozymes confer a selective survival advantage on CD4+ T cells in vitro

To date, a selective advantage of cells expressing anti-HIV ribozymes has not been shown. This study was undertaken to determine whether such a selective advantage can be demonstrated in vitro. A retroviral vector coding for a hairpin ribozyme targeting the HIV 5′LTR and for the low affinity nerve growth factor receptor (LNGF-RΔ) was designed. Since we demonstrated by RT-PCR that the amount of ribozyme transcripts was highly correlated with the level of surface LNGF-RΔ expression, the vector was utilized to assess ribozyme expression by flow cytometry. Transduced Hut78 and primary CD4+ T cells were purified and subsequently mixed with unmodified cells. After HIV challenge the percentage of ribozyme expressing cells in the cell mixture was monitored by flow cytometry. Twenty-one days after HIV infection the proportion of ribozyme expressing CD4+ T cells was 2.6 times higher in comparison to cells with the control vector. CD4+ T cells with a strong ribozyme expression conferred a 7.4-fold selective advantage at day 21 and a 11.7-fold at day 28. For Hut78 cells a selective advantage was detected exclusively for strongly ribozyme expressing cells. As a mechanism underlying the selective advantage an inhibition of HIV induced apoptosis was shown. These results demonstrate that anti-HIV ribozymes are able to confer a selective survival advantage and indicate that the protective effect is dependent on the amount of ribozyme expression.

Effective reversal of a transformed phenotype by retrovirus-mediated transfer of a ribozyme directed against mutant N-ras

A hammerhead ribozyme directed against oncogenic N-ras (N13-ras) was introduced into a retroviral vector and its activity evaluated in vitro and in cell lines. The catalytic efficiency of the ribozyme embedded within a 2618 nucleotides in vitro-generated transcript was not significantly affected by the length of non-base pairing flanking sequences. A sensitive assay based on N-ras/luciferase fusion transcripts as a reporter system was used to assess ribozyme activity in mammalian cells. More than 95% reduction in luciferase activity was observed in cells transduced with a retrovirus containing the active form of the ribozyme, whereas no significant reduction was observed with the inactive form of the same ribozyme. In order to assay the activity of the retrovirally encoded ribozyme in a biological setting, the IL-3-dependent cell line TF-1 was transformed with N13-ras. Expression of N13-ras in these cells induced factor-independent colony growth and a dose-dependent proliferative response to erythropoietin (Epo). Retrovirus-mediated expression of the active form of the ribozyme in these cells restored factor-dependent colony growth and abolished the proliferative response to Epo. The reversion of the transformed phenotype correlated with a reduction in the amount of N13-ras mRNA.

Synthesis and properties of hammerhead ribozymes stabilized against nucleases by different 2′-modifications: methoxyethoxy-, fluoro- and amino groups

Abstract 2′-modified ribonucleosides (2′-methoxyethoxy- O, 2′-fluoro-2′-deoxypyrimidines O and 2′-amino-2′-deoxyuridines □) were introduced into hammerhead ribozymes. Kinetics and stability assays were performed. The lipophilicity characteristics of these 2′-modifications were investigated measuring partition coefficients and HPLC retention times.

Modified Hammerhead Ribozymes as Potential Therapeutics

Abstract Hammerhead ribozymes were modified in the 2′-position by flouro-, amino-, deoxy-, O-methyl- and methoxyethoxy-groups to stabilize against degradations. They were tested for their ability to cut specifically oncogenic N-ras RNA in vitro. Exogenous delivery by lipofection as well as viral vector mediated transfection showed comparable results in reducing N-ras mRNA.