The major focus of this laboratory is control of gene expression by antisense oligonucleotides and RNA.
Research Synopsis:
Antisense oligonucleotides represent a new class of compounds that are emerging as highly specific therapeutics.
They interact with mRNA in a sequence specific fashion downregulating the expression of the gene from which the RNA
has been transcribed. In a novel approach, we have used oligonucleotides to modulate gene expression by modifying
alternative splicing of targeted pre-mRNA. The importance of this approach is underscored by recent findings that up
to 70% of all human genes are alternatively spliced. Thus, sequence specific modification of splicing by splice
switching oligonucleotides (SSO) promises to be useful in a broad range of therapeutic indications with thousands of
genes as potential targets. This notion is illustrated by the work in this laboratory, which showed that modification
of alternative splicing of Bcl-x, a gene over-expressed in prostate cancer cells, leads to cell apoptosis and may
provide gene specific treatment for prostate and other cancers. Furthermore, by targeting the SSOs against aberrant
splice sites generated by genetic mutations we were able to reverse aberrant and restore correct splicing and therefore
increase the levels of correctly spliced pre-mRNAs in cellular models of ß-thalassemia, cystic fibrosis and
Duchenne muscular dystrophy. The work on thalassemia and muscular dystrophy progressed to animal models of these
disorders and to ex vivo treatment of erythroid cells from thalassemia patients. As an extension of the above
work, we developed methods that take advantage of lentiviruses as vectors to provide gene therapy alternatives to
oligonucleotide treatment. These methods have been successfully applied to thalassemic mutants.
(See Figure)
Members of the laboratory become knowledgeable in RNA processing and splicing and macromolecular drug d
elivery and oligonucleotide technology. They acquire expertise in a variety of techniques such as site specific
mutagenesis, PCR and RT-PCR, transient and stable gene expression in cultured cells, delivery of antisense
oligonucleotides and vectors for intracellular activity in cell culture and in animal models.
Recent Publications:
Williams, T., and Kole, R. (2006) Analysis of prostate specific membrane antigen splice variants in LnCap cells. Oligonucleotides. [In press]
Kole, R., Vacek, M., and Williams, T. (2004) Modification of alternative splicing by antisense therapeutics. Oligonucleotides 14(1): 65-74. Abstract
Bristow, C.L., Mercatante, D.R., and Kole, R. (2003) HIV-1 preferentially binds receptors co-patched with cell surface elastase. Blood 102(13): 4479-86. Abstract
Vacek, M., Ma, H., Gemignani, F., Lacerra, G., Kafri, T., and Kole, R. (2003) High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA. Blood 101(1): 104-11. Abstract
Sazani, P., Gemignani, F., Kang, S-H., Maier, M.M., Manoharan, M., Persmark, M., Bortner D., and Kole, R. (2002). Functional analysis of modified oligonucleotides in vivo. Systemic delivery, biodistribution and antisense effects. Nature Biotech 20(12): 1228-1233. Abstract
Mercatante, D.R., Mohler, J.L., and Kole. R. (2002) Cellular response to an antisense-mediated shift of Bcl-x pre-mRNA splicing and antineoplastic agents. J Biol Chem277(51): 49374-49382. Abstract
