Download 311 Human Genetics

311 Human Genetics
Fall 2006
Lecture: RNA interference (RNAi)
References:
(1) Tuschl, T. and Borkhardt, A. (2002) Small interfering RNAs. Molecular
interventions 2, 158-167.
(2) Hannon, G. J. And Rossi, J. J. (2004) Unlocking the potential of the human genome
with RNA interference. Nature 431, 371-378.
(3) Gaur, R. K. (2006) RNA interference: a potential therapeutic tool for silencing
splice isoforms linked to human diseases. Biotechniques. Apr;Suppl:15-22.
Lecture outline:
1. Discovery of RNAi
2. RNAi mechanism
3. RNAi targeted therapies
a. genetic diseases: point mutations, triplet expansions
b. splicing mutations
c. cancer genetic therapies: BCR-ABL
d. HIV: CCR5 coreceptor
4. Issues of RNAi therapies
a. specific targeting
b. targeting
c. interfering with normal regulation
Lecture:
1. Discovery of RNAi
a. Fire et al. 1998 showed that gene silencing in C. elegans (nematode) could be
triggered by double-stranded RNA.
b. Led to discovery of C. elegans genes involved in RNAi. Process found to be
conserved in Drosophila, plants, fungi.
c. Enzyme “dicer” was found to be responsible for generating small interfering RNAs.
d. Pathways for RNAi action vary slightly between organisms. Many involve
downstream complex of one strand of miRNA (microRNA) targeting RNA cleavage of
mRNA in RISC complex (RNA-induced silencing complex).
2006 Nobel Prize in Physiology and Medicine awarded to Andrew Fire and Craig Mello
for discovery of gene silencing mediated by RNAi.
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2. RNAi mechanism:
a. Long dsRNA and miRNA precursors are processed to siRNA/miRNA duplexes by
Dicer.
b. Short dsRNAs are unwound and assembled into RISCs which direct RNA cleavage,
mediate translational repression or induce chromatin modification.
3. RNAi targeted therapies
Potential for treating a variety of diseases: human single gene disorders, cancers, viral
and parasitic diseases.
Can “practice” therapies in human cell lines or animal models to test feasibility.
Unlike stem cell therapies, less likelihood of triggering an immune response.
a. Genetic diseases: use RNAi to target point mutations, triplet expansions.
i) ALS: amyotrophic lateral schlerosis
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Lou Gehrig’s disease
 Caused by mutations in SOD1 gene; encodes Cu, Zn superoxide dismutase
 Many SOD1 mutations in ALS are single nucleotide changes.
Ding et al. were able to design an siRNA that targets only the mutant SOD1 allele,
leaving the wild type allele intact.
ii) SBMA: spinobulbular muscular atrophy
CAG triplet expansion disease
Caplan et al. used siRNA in human tissue culture cells to target the CAG-expanded
mRNA for SBMA. Reduces toxic effects of polyglutamine protein encoded by triplet
expansion.
Can possibly be applied to other triplet expansion diseases such as HD.
b. splicing mutations
may contribute to genetic diseases and cancer
HGH:



human growth hormone gene
Contains 5 exons, 4 introns
Normal splicing produces mRNA for 22 kDa HGH protein
In some mutant forms due to splice site mutations, exon 3 is skipped in splicing to
produce a 17.5 kDa protein
17.5 kDa protein is associated with the autosomal dominant HGH protein deficiency.
Design siRNA to target variants with exon 2-exon 4 junction to selectively target and
destroy 17.5 kDa mRNA isoform.
c. Cancer genetic therapies
Specific cancer genes and mutations can be targeted by RNAi.
d. BCR-ABL
A common mutation associated with CML (chronic myeloid leukemia) is the
translocation of the BCR and ABL oncogenes t(9;22). The BCR gene from chromosome
22 and ABL gene on chromosome 9 become fused creating an oncogenic BCR-ABL
hybrid gene.
The chimeric oncoprotein is an overactive tyrosine kinase. The BCR-ABL tyrosine
kinase is the basis for a targeted therapy (marketed as Gleevac), currently used
successfully to treat CML and a few other cancers. Resistance to Gleevac could
potentially be treated using an RNAi approach.
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siRNA treatment reduces expression of the BCR-ABL mRNA, followed by reduction of
the BCR-ABL oncoprotein, leading to apoptosis in leukemic cells.
d. HIV
possible targets: cellular proteins such as CCR5 co-receptor; HIV proteins
Rapid evolution of HIV virus makes targeting of virus consistently to be difficult.
Therefore targeting of cellular factors is better.
Individuals with homozygous Δ32 allele of CCR5 are resistant to AIDS infection.
Heterozygotes show delayed progression to AIDS. Receptor not essential for immune
function.
Therefore a strategy of RNAi is to down-regulate levels of CCR5; reduces infectivity of
HIV virus. However, virus can still infect other T-cells with different coreceptor that is
essential for immune function and can’t be targeted for therapy.
4. Issues of RNAi therapies
a. specific targeting
 need DNA sequences of normal vs. wild type sequences
 use computer algorithms to design
 siRNAs
 may need to expt. test several possibilities
b. delivery
 a problem for RNAi and existing gene therapy strategies
 transfection methods (electroporation, injection, or chemical treatment)
 different efficiencies for different methods and not all cell types easily transfected
 therapy can be directly delivered as siRNA or can be expressed from a vector.
 Viral delivery vectors may be cell-type or tissue specific or may lead to immune
response in the whole organism.
c. Therapy may interfere with normal gene regulatory pathways.
Endogenous RNAis may regulate many cell processes; most of these are uncharacterized
The human genome project revealed there are ~200 microRNAs in the human genome
and >1500 antisense RNAs; the functions of these are mostly not known. In total, these
comprise half of all human RNA genes.
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