Transcript Interfering RNA

Patenting Interfering RNA
John LeGuyader – SPE Art Unit 1635
(571) 272-0760
[email protected]
Interfering RNA
Glossary of Terms
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RNAi – RNA interference
dsRNA – double stranded RNA
Dicer – RNase endonuclease
siRNA – small interfering RNA, double stranded, 21-23
nucleotides
shRNA – short hairpin RNA (doubled stranded by
virtue of a ssRNA folding back on itself)
ssRNA – single stranded RNA
RISC – RNA-induced silencing complex
Helicase – unwinds siRNA and RNase endonuclease
RNAi - Mechanism of Action
• Introducing long double stranded RNA leads to
sequence-specific degradation of homologous gene
transcripts
• Long double stranded RNA metabolized to small 21-23nucleotide siRNAs by endogenous RNase Dicer
• siRNAs bind to protein complex RISC with dual
function helicase – unwinding and RNase activity
• Unwinds siRNA allowing antisense strand to bind to
target
• Hydrolyses of target by endonuclease activity of
helicase
Antisense
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ssDNA - 5 deoxynucleotides
8-30 mer
Endonuclease - ribonuclease
Antisense binds homologous
RNA target
• RNAse H endonuclease
• attacks DNA/RNA duplex
• Sequence-dependent
degradation of cognate mRNA
RNAi
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siRNA: ds- or ssRNA
21 mer
Endonuclease - ribonuclease
Antisense binds homologous
RNA target
• Helicase unwinds/endonuclease
• attacks RNA/RNA duplex
• Sequence-dependent degradation
of cognate mRNA
RNAi Patentability issues
• Consider a broad claim to:
An siRNA that inhibits expression of a
nucleic acid encoding protein X.
• Can also be claimed more broadly as
short interfering nucleic acid.
RNAi Patentability Issues
35 U.S.C. 101 – Utility
• Credible/Specific/Substantial/Well Established
• Used to routinely investigate gene function in a high
throughput fashion or to modulate gene expression in
human diseases (see Chi et al. (PNAS) 100(11):63436346, 2003)
• Some knowledge of gene function required
• Enough to warrant target inhibition
RNAi Patentability Issues
35 U.S.C. 101 – Utility
• If no function for target nucleic acid (protein or
regulatory) is shown or was known:
– RNAi would likely lack utility
– also raises possible enablement (how to use) and/or written
description issues
– probe function alone for target not sufficient to provide utility
for RNAi
RNAi Patentability Issues
35 U.S.C. 112, first paragraph, Enablement
• high in vivo unpredictability due to general lack
of knowledge regarding efficacy and in vivo
target site determination, and delivery issues
• to date only one human antisense with FDA
approval
RNAi Predictability
• Highly dependent on target position
• Screening required to identify accessible target regions
• Antisense targets can also be RNAi targets but with
exceptions, e.g. intron targets not active for RNAi
• Small mismatches (1-2 nts) can be tolerated
• Long dsRNAs cause severe sequence-non-specific
effects
– induces apoptosis from shut down of translation
– RNAi of about 21nts required to evade effects
• Little in vivo efficacy to date
RNAi Patentability Issues
35 U.S.C. 112, first paragraph, Written Description
• adequate description for broad/generic claims relates to
what is known and/or disclosed regarding target site
accessibility for the gene at issue
• the example claim defines the invention in purely
functional terms and lacks structural correlates
• RNAi described only by function may lack written
description
RNAi Patentability Issues
35 U.S.C. 112, first paragraph, Written Description
• The Analysis:
– identify all disclosed relevant distinguishing characteristics as they relate to
the scope and content of the claims
– identify any disclosed structure/function relationships
• what target regions are accessible for RNAi and provide for inhibition
• showing of antisense targets across mRNA may be sufficient, but not
all antisense targets are open to siRNA
• intron targets may not be active for siRNA but may be for antisense
– identify all elements claimed and their support in the description
– identify species explicitly or implicitly disclosed
– reconcile with the level of skill in the art
RNAi Patentability Issues
35 U.S.C. 112, first paragraph, Written Description
• A broad genus of siRNAs inhibiting expression of
a nucleic acid encoding a protein may not be
described by the sequence of a gene alone since
the genus reads on targeting many different
nucleic acids.
• Description of genus sufficient to comply with
written description requires description of a
representative number of species
• In the case of a small genus covering a limited
defined target, one species may be respresentative
RNAi Patentability Issues
35 U.S.C. 112, first paragraph, Written Description
• Written Description Conclusions:
– Broad claims to siRNAs inhibiting expression of a
nucleic acid encoding a protein may lack an adequate
written description since the claim reads on targeting
many different nucleic acids.
– Analysis turns on what is shown in the specification
and what was known about the various versions of the
gene at the time of filing.
– Provide evidence RNAi targets shown functionally
correlate with targeting other versions of the gene.
– The more you show and/or is known, the more you can
possibly claim.
% Inhibition
Gene Walk
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RNAi/Antisense Target - 5' to 3'
Gene Walk Conclusions
• Probability of finding an individual functional RNAi is high
– A broad claim to “An isolated RNAi that inhibits the expression of
human gene X.” may be enabled by providing the sequence for gene X
and gene walk data (no magic number)
• Predictability of any single RNAi being effective is low
– claims to specific RNAi sequence may require evidence of function
• The current state of predictability for RNAi may support that
breadth/scope claimed is enabled
– but this may also raise prior art issues depending on what was known at
the time of filing
RNAi Patentability Issues
35 U.S.C. 102 – Novelty/Anticipation
• Anticipation of specific RNAi
– must be explicitly taught in the prior art for
anticipation to be applicable.
RNAi Patentability Issues
35 U.S.C. 103 - Obviousness
• Expect an obviousness rejection against broad RNAi claims to
known genes if the prior art suggested inhibiting the gene by
nucleic acid-based methods or other means and the gene
sequence was known.
• The current knowledge and level of skill in the art is high such
that one of ordinary skill in the art would expect at least an
RNAi against a known gene, absent evidence to the contrary.
• Narrow claims to specific RNAi sequences may be free of the
art, where there would be no motivation to modify the prior art
to achieve the specific RNAi sequence claimed.
RNAi Patentability Issues
35 U.S.C. 103 - Obviousness
• Motivation
• used to routinely investigate gene function in a high
throughput fashion or to modulate gene expression in
human diseases (see Chi et al. (PNAS) 100(11):63436346, 2003)
• target identified in the prior art as desirable for silencing
(disease gene, virus), and which has been inhibited in cells
• neither necessarily identifies any specific RNAi sequence
RNAi Patentability Issues
35 U.S.C. 103 - Obviousness
• Expectation of Success
• expectation of RNAi gene silencing highly likely for target
sites identified as accessible to antisense inhibition (see
Vickers et al. (J. Biol. Chem.) 278: 7108-7118, 2003)
• low expectation of success for in vivo applications
• low expectation for specific target sequences which are not
identified
• identification of some sequence as appropriate target
should be provided, e.g. known antisense target
Case Law
• Antisense-specific:
– Enzo Biochem Inc. v. Calgene Inc., 52 USPQ2d 1129
(CAFC 1999)
– enablement - antisense highly unpredictable
– the decision is based on patents with effective filing
dates of at least 1989 and the technology at that time
– decision does not necessarily determine the outcome for
examination of antisense patent applications recently
filed because current knowledge and level of skill in the
art is high (antisense has progressed as a technology
since 1989)
Recommendations
• Claim functional RNAi by specific sequence.
• List results of “gene walk”
– showing activity of each RNAi
– “gene walk” data may provide representative number of
species for broad scope of a generic claim, but there is
no magic number
Recommendations
• Provide objective evidence that in vitro results are
representative of in vivo applicability.
• Respond to examiner-cited unpredictable factors with
objective evidence to the contrary.
• Expert opinions are more favorably viewed when
supported using objective evidence.
• Provide objective evidence that a particular animal model
is generally accepted as representative of disease or
methods of treating, particularly for humans.
• Objective evidence includes arguments, case law, journal
articles, and experimental data and comparisons
commensurate with the disclosure as filed.
RNAi
Questions?