DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 1-15-26.
Claims 282, 285-310 are pending in the instant application.
Election/Restrictions
Claims 1-3, 15, 36, 41, 43, 76, 125, 127, 139, 154, 159, 219, 220, 222, 272-275, 277, 278, and 280 have been canceled and are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions of species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 1-15-26.
Applicant’s election without traverse of Group IV, claims 282, 285-310, in the reply filed on 1-15-26 is acknowledged.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 282, 285-291, 293-310 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling for in vitro efficacy data. in HeLa cells transfected (using RNAiMax) with Di-branched RNA oligonucleotides, for the treatment of primary cortical mouse neurons in vitro with Di-branched RNA oligonucleotides, and for the delivery of siRNA molecules comprising the linkages represented by Formula I via intrastriatal, intrathecal, lumbar intrathecal, or intracerebroventricular administration of Di-branched RNA oligonucleotides, does not reasonably enable methods of delivering siRNA molecules to the central nervous system of a subject, methods of delivering an siRNA molecule for allele-specific silencing of any gene in a subject in need thereof, and/or methods of delivering siRNA molecules for silencing any mRNA in a subject in need thereof comprising the administration, by any route, of the RNA oligonucleotides instantly claimed, and further whereby adequate delivery and therapeutic effects have been provided in any subject.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
The following factors have been considered in determining that the specification does not enable the skilled artisan to make and/or use the invention over the broad scope claimed.
The breadth of the claims:
The claims are drawn to methods of delivering an siRNA molecule to the central nervous system of a subject, methods of delivering an siRNA molecule for allele-specific silencing of any gene in a subject in need thereof, and methods of delivering an siRNA molecule for silencing any mRNA in a subject in need thereof, which methods comprise administering to the subject one or more double-stranded, chemically modified oligonucleotide compounds, each comprising an antisense strand having complementarity to a target mRNA molecule and a sense strand having complementarity to the antisense strand, wherein at least one of the antisense strand and the sense strand comprises a modified intersubunit linkage represented by Formula I:
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wherein each of the antisense strand and the sense strand is, independently, from 10 to 50 nucleotides in length, from 15 to 25 nucleotides in length, from 20 to 21 nucleotides in length, 15 or 16 nucleotides in length, 21 nucleotides in length, or 16 nucleotides in length, and which administration optionally comprises intrathecal, intrastriatal, or intracerebroventricular injection.
Teachings in the art and in the specification.
Teachings in the art:
Roberts et al (Nature Rev., Drug Discovery, Vol. 19, pages 673-694 (2020)) teaches on page 673 that “achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation.”
Kobelt et al (Cancer Gene Therapy in Gene Therapy of Cancer: Methods and Protocols, Methods in Molecular Biology, Vol. 2521, pages 1-15 (Springer Nature 2022)) teach that limitations to cancer gene therapy relate to limitations in gene transfer efficiency (see esp. pages 3-4).
In addition, Osborn et al (Nucleic Acid Therapeutics, Vol. 28, No. 3, pages 128-136 (2018)) state the following about challenges to siRNA delivery on page 128:
…The primary challenge facing the clinical development of small interfering RNAs (siRNA) has been overcoming barriers that impede in vivo delivery. siRNAs are large, polyanionic macromolecules with intrinsically poor pharmacological properties. Unmodified siRNAs have a half-life of less than 5 min in circulation, and they do not permeate intact cellular membranes…
Damase et al (Frontiers in Bioengineering and Biotechnology, Vol. 9, Article 628137, pages 1-24 (2021)) on page 13 also address the challenges of using RNA-based drugs:
Targeted delivery is a major hurdle for effective RNA therapeutics, a hurdle that must be overcome to broaden the application of clinical translation of this type of therapeutic. …There is a need for novel delivery vehicles that will deliver the RNA drug to the site of therapeutic action facilitating the entry of the RNA drug into the cytoplasm where it may exert its effect…
Teachings in the specification:
The specification teaches the following
[0189] FIG. 2A shows a representative example for preparing a monomer for the
modified phosphinate-containing oligonucleotides provided herein.
[0190] FIG. 2B shows a representative example for preparing another monomer for the modified phosphinate-containing oligonucleotides provided herein.
[0191] FIG. 2C shows representative example for preparing a modified phosphinate- containing oligonucleotide provided herein.
[0192] FIG. 3A shows a representative example for preparing a monomer for the
modified phosphonate-containing oligonucleotides provided herein.
[0193] FIG. 3B shows representative example for preparing a modified phosphonate- containing oligonucleotide provided herein.
[0194] FIG. 4 provides a representative method for preparing the oligonucleotides provided herein on solid support.
[0195] FIG. 5 shows a method for preparing a vinylphosphinate-modified
oligonucleotide.
[0196] FIG. 6 shows a representative example for preparing a monomer for the
synthesis of the oligonucleotides provided herein.
[0197] FIG. 7 shows a representative example for preparing a monomer for the
synthesis of the modified oligonucleotides provided herein.
[0198] FIG. 8 illustrates the method for preparing the modified oligonucleotides
provided herein.
[0199] FIG. 9A illustrates the impact of internal-VP modification on siRNA thermal
stability.
[0200] FIG. 9B illustrates the impact of internal-VP modification on duplex RNA
thermal stability.
[0201] FIG. 10A shows RNA stability in a digestion test by SVPD (3'
5' exonuclease) x4 condition for RNA having PO, VP, and PS linkers.
[0202] FIG. 10B shows RNA stability in a digestion test by SVPDE (3' 5' exonuclease) x10 condition for RNA having PO, VP, and PS linkers.
[0203] FIG. 10C shows RNA stability in a digestion test by SVPDE (3'
5' exonuclease) for VP/PS mixed sequences.
[0204] FIG. 11 illustrates the effect of adding a mismatch in the siRNA sequence to improve allelic discrimination without impairing silencing of the mutant allele.
[0205] FIG. 12 illustrates the silencing efficacy of VP-modified siRNA.
[0206] FIG. 13 depicts a method for preparing oligonucleotides having a vinyl
5 phosphonate modified intersubunit linkage described herein.
[0207] FIG. 14 illustrates the sequences of vinyl phosphonate modified
oligonucleotides synthesized. Antisense strands are depicted 5' to 3', with the SNP site in red and the mismatch in blue.
[0208] FIG. 15 is a schematic representation of hsiRNA antisense scaffolds aligned to the HTT sequence surrounding SNP site rs362273, wherein the green box indicates the position of the SNP site.
[029] FIG. 16 depicts the change in mRNA expression when standard siRNA is used versus VP-modified siRNA.
[0210] FIG. 17 shows on or off-target HTT-mRNA knock down with control and
15 VP-modified siRNA.
[0211] FIG. 18 shows the structure of Di-hsiRNAs. Black - 2'-O-methyl, grey - 2°-
fluoro, red dash - phosphorothioate bond, linker - tetraethylene glycol. Di-hsiRNAs are two asymmetric siRNAs attached through the linker at the 3' ends of the sense strand. Hybridization to the longer antisense strand creates protruding single stranded fully phosphorthioated regions, essential for tissue distribution, cellular uptake and efficacy. The structures presented utilize teg linger of four monomers. The chemical identity of the linker can be modified without the impact on efficacy. It can be adjusted by length, chemical composition (fully carbon), saturation or the addition of chemical targeting ligands.
[0212] FIG. 19 shows a chemical synthesis, purification and quality control of Di-
branched siRNAs.
[0213] FIG. 20 shows HPLC and quality control of compounds produced by the
method depicted in FIG. 19. Three major products were identified by mass spectrometry as sense strand with TEG (tetraethylene glycol) linker, di-branched oligo and Vit-D (calciferol) conjugate. All products where purified by HPLC and tested in vivo independently. Only Di- 30 branched oligo is characterized by unprecedented tissue distribution and efficacy, indicating that branching structure is essential for tissue retention and distribution.
[0214] FIG. 21 shows mass spectrometry confirming the mass of the Di-branched
oligonucleotide. The observed mass of 11683 corresponds to two sense strands attached through the TEG linker by the 3' ends.
[0215] FIG. 22 shows a synthesis of a branched oligonucleotide using alternative
5 chemical routes.
[0216] FIG. 23 shows exemplary amidite linkers, spacers and branching moieties.
[0217] FIG. 24 shows oligonucleotide branching motifs. The double-helices
represented oligonucleotides. The combination of different linkers, spacer and branching points allows generation of a wide diversity of branched hsiRNA structures.
[0218] FIG. 25 shows structurally diverse branched oligonucleotides.
[0219] FIG. 26 shows an asymmetric compound of the invention having four single-stranded phosphorothioate regions.
[0220] FIGS. 27A-27C show in vitro efficacy data. (Fig. 27A) HeLa cells were
transfected (using RNAiMax) with Di-branched oligo at concentrations shown for 72 hours.
(Fig. 27B) Primary cortical mouse neurons were treated with Di-branched oligo at
concentrations shown for 1 week. mRNA was measured using Affymetrix Quantigene 2.0.
(Fig. 27C) HeLa cells were treated passively (no formulation) with Di-siRNA oligo at concentrations shown for 1 week.
[0221] FIGS. 28A-28B show brain distribution of Di-siRNA or TEG only after 48
hours following intra-striatal injection. Intrastriatal injection of 2 nmols of (Fig. 28A) Di- branched oligo (4 nmols of corresponding antisense strand) or (Fig. 28B) TEG-oligo only. N=2 mice per conjugate. Brains collected 48 hours later and stained with Dapi (nuclei, blue). Red - oligo. The left side of brain in (Fig. 28A) appears bright red, whereas the left side of the brain in (Fig. 28B) only faintly red.
[0222] FIG. 29 shows that a single injection of Di-siRNA was detected both
ipsilateral and contralateral to the injection site.
[0223] FIGS. 30A-30B shows Di-hsiRNA wide distribution and efficacy in mouse
brain.
(Fig. 30A) Robust Htt mRNA silencing in both Cortex and Striatum 7 days after single IS injection (25 µg), QuantiGene®. (Fig. 30B) Levels of hsiRNA accumulation in tissues 7 days after injection (PNA assay).
[0224] FIGS. 31A-31C show wide distribution and efficacy throughout the spinal
cord following bolus intrathecal injection of Di-hsiRNA. Intrathecal injection in lumbar of 3 nmols Di-branched Oligo (6 nmols of corresponding antisense HTT strand).
(Fig. 31A) Robust Htt mRNA silencing in all region of spinal cord, 7 days, n-6. Animals sacrificed 7 days post-injection. Tissue punches taken from cervical, thoracic and lumbar regions of spinal cord. mRNA was quantified using Affymetrix Quantigene 2.0 as per Coles et al. 2015. ….
(Fig. 31B) Animals were injected lumbar IT with 75 µg of Cy3-Chol- hsiRNA, Cy-Di-hsiRNA. Chol-hsiRNAs shows steep gradient of diffusion from outside to
inside of spinal cord. Di-hsiRNAs shows wide distribution throughout the cord (all regions). Leica 10x (20mm bar). Image of Di-branched oligo in cervical region of spinal cord 48 hours after intrathecal injection. Red = oligo, Blue = Dapi.
(Fig. 31C) Image of Di-branched oligo in liver 48 hours after intrathecal injection. Red = oligo, Blue = Dapi.
[0225] FIGS. 32A-32C show branched oligonucleotides of the invention,
(Fig. 32A)
formed by annealing three oligonucleotides. The longer linking oligonucleotides may
comprise a cleavable region in the form of unmodified RNA, DNA or UNA;
(Fig. 32B) asymmetrical branched oligonucleotides with 3' and 5' linkages to the linkers or spaces described previously. This can be applied the 3' and 5' ends of the sense strand or the antisense strands or a combination thereof; (Fig. 32C) branched oligonucleotides made up of three separate strands. The long dual sense strand can be synthesized with 3' phosphoramidites and 5' phosphoramidites to allow for 3'-3' adjacent or 5'-5' adjacent ends.
[0226] FIG. 33 shows branched oligonucleotides of the invention with conjugated
bioactive moieties.
[0227] FIG. 34 shows the relationship between phosphorothioate content and stereoselectivity.
[0228] FIG. 35 depicts exemplary hydrophobic moieties.
[0229] FIG. 36 depicts exemplary internucleotide linkages.
[0230] FIG. 37 depicts exemplary internucleotide backbone linkages.
[0231] FIG. 38 depicts exemplary sugar modifications.
[0232] FIGS. 39A-39C depict Di-FM-hsiRNA.
(Fig. 39A) Chemical composition of the four sub-products created from VitD-FM-hsiRNA synthesis and crude reverse phase analytical HPLC of the original chemical synthesis. (Fig. 39B) Efficacy of sub-products in HeLa cells after lipid mediated delivery of hsiRNA. Cells were treated for 72 hours. mRNA was measured using QuantiGene 2.0 kit (Affymetrix).
(Fig. 39C) A single, unilateral intrastriatal injection (25 µg) of each hsiRNA sub-product. Images taken 48 hours after injection.
[0233] FIGS. 40A-40B show that Di-HTT-Cy3 does not effectively induce silencing in the liver or kidneys following intrastriatal injection.
FIG. 40A depicts a scatter dot plot showing Htt mRNA expression in the liver one-week post intrastriatal injection of Di-HTT- Cy3 compared to a negative control (aCSF).
FIG. 40B depicts a scatter dot plot showing Htt mRNA expression in the kidney one-week post intrastriatal injection of Di-HTT-Cy3…
[0234] FIGS. 41A-41B shows that Di-HTT effectively silences HTT gene expression in both the striatum and the cortex following intrastriatal injection and that Di-HTT-Cy3 is slightly more efficacious than Di-HTT (unlabeled).
FIG. 41A depicts a scatter dot plot showing Htt mRNA expression in the striatum one-week post intrastriatal injection of Di-HTT, Di-HTT-Cy3, or two negative controls (aCSF or Di-NTC).
FIG. 41B depicts a scatter dot plot showing Htt mRNA expression in the cortex one-week post intrastriatal injection of Di-HTT, Di-HTT-Cy3, or two negative controls (aCSF or Di-NTC).
[0235] FIG. 42 depicts a scatter dot plot measuring Di-HTT-Cy3 levels in the
striatum and cortex. The plot shows that significant levels of Di-HTT-Cy3 are still detectable two weeks post intrastriatal injection.
[
0236] FIGS. 43A-43B show that Di-HTT-Cy3 effectively silences HTT mRNA and
protein expression in both the striatum and the cortex two weeks post intrastriatal injection.
FIG. 43A depicts a scatter dot plot measuring Htt mRNA levels in the striatum and cortex two weeks post injection.
FIG. 43B depicts a scatter dot plot measuring Htt protein levels in the striatum and cortex two weeks post injection.
[0237] FIGS. 44A-44B show that high dose Di-HTT-Cy3 treatment does not cause significant toxicity in vivo but does lead to significant gliosis in vivo two weeks post intrastriatal injection.
FIG. 44A depicts a scatter dot plot measuring DARPP32 signal in the striatum and cortex two weeks after injection with Di-HTT-Cy3 or aCSF. FIG. 44B depicts a scatter dot plot measuring GFAP protein levels in the striatum and cortex two weeks after injection with Di-HTT-Cy3 or aCSF.
[0238] FIG. 45 depicts fluorescent imaging showing that intrathecal injection of Di-HTT-Cy3 results in robust and even distribution throughout the spinal cord.
[0239] FIG. 46 depicts a merged fluorescent image of FIG. 45 (zoom of spinal cord). Blue-nuclei, red-Di-HTT-Cy3.
[0240] FIGS. 47A-47C shows the widespread distribution of Di-HTT-Cy3 48 hours post intracerebroventricular injection.
FIG. 47A depicts fluorescent imaging of sections of the striatum, cortex, and cerebellum.
FIG. 47B depicts brightfield images of the whole brain injected with control (aCSF) or Di-HTT-Cy3. FIG. 47C depicts a fluorescent image of a whole brain section 48 hours after Di-HTT-Cy3 injection.
[0241] FIG. 48 shows that Di-HTT-Cy3 accumulates in multiple brain regions two
weeks post intracerebroventricular injection. A scatter dot plot measures the level of Di-HTT- Cy3 in multiple areas of the brain.
[0242] FIG. 49A shows that Di-HTT-Cy3 induces Htt gene silencing in multiple
regions of the brain two weeks post intracerebroventricular injection compared to a negative control injection (aCSF). A scatter dot plot measures Htt mRNA levels in multiple areas of the brain.
FIG. 49B shows that Di-HTT-Cy3 induces Htt silencing in multiple regions of the
brain two weeks post intracerebroventricular injection compared to a negative control injection (aCSF). A scatter dot plot measures Htt protein levels in multiple areas of the brain.
[0243] FIG. 50 shows that intracerebroventricular injection of high dose Di-HTT- Cy3 causes minor toxicity in vivo. A scatter dot plot measures DARPP32 signal in multiple regions of the brain following Di-HTT-Cy3 of aCSF injection.
[0244] FIG. 51 shows that intracerebroventricular injection of high dose Di-HTT- Cy3 causes significant gliosis in vivo. A scatter dot plot measures DARPP32 signal in multiple regions of the brain following Di-HTT-Cy3 of aCSF injection.
[0245] FIG. 52 shows that Di-HTT-Cy3 is distributed to multiple organs following
intravenous injection. Fluorescent images depict Di-HTT-Cy3 levels in the heart, kidney, adrenal gland, and spleen following intravenous injection of Di-HTT-Cy3 or a negative control (PBS).
[0246] FIG. 53 shows that Di-HTT-Cy3 accumulates in multiple organs following intravenous injection. A scatter dot plot measures the levels of Di-HTT-Cy3 in multiple tissues.
[0247] FIG. 54 illustrates the structures of hsiRNA and fully metabolized (FM)
hsiRNA.
[0248] FIGS. 55A-55B show that full metabolic stabilization of hsiRNAs results in
more efficacious gene silencing following intrastriatal injection of hsiRNA HTT or FM-hsiRNA HTT.
FIG. 55A depicts a scatter dot plot measuring HTT mRNA levels up to 12
days after intrastriatal injection.
FIG. 55B depicts a scatter dot plot measuring HTT mRNA levels up to 28 days after intrastriatal injection.
[
0249] FIG. 56 depicts the chemical diversity of single stranded fully modified
oligonucleotides. The single stranded oligonucleotides can consist of gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, or PNAs.
[0250] FIG. 57 depicts Di-HTT with a TEG phosphoramidate linker.
[0251] FIG. 58 depicts Di-HTT with a TEG di-phosphate linker.
[0252] FIG. 59 depicts variations of Di-HTT with either two oligonucleotide
branches or four oligonucleotide branches.
[0253] FIG. 60 depicts another variant of Di-HTT of a structure with two
oligonucleotide branches and R2 attached to the linker.
[0254] FIG. 61 depicts a first strategy for the incorporation of a hydrophobic moiety into the branched oligonucleotide structures.
[0255] FIG. 62 depicts a second strategy for the incorporation of a hydrophobic moiety into the branched oligonucleotide structures.
[0256] FIG. 63 depicts a third strategy for the incorporation of a hydrophobic moiety into the branched oligonucleotide structures.
[0257] FIG. 64 depicts in vitro silencing efficacy of target mRNA with siRNA
duplexes containing ex-NA intersubunit linkages at various positions.
[0258] FIG. 65 depicts the ex-NA (2'O-Methyl) phosphoramidite synthesis scheme.
[0259] FIG. 66 depicts the ex-NA (2'-Fluoro) phosphoramidite synthesis scheme.
[0260] FIG. 67 depicts the coupling of ex-NA phosphoramidite on solid support.
[Emphases added][Citations omitted].
The examples provided in the instant specification, of in vitro efficacy data. in HeLa cells transfected with Di-branched RNA oligonucleotides, for the treatment of primary cortical mouse neurons in vitro with Di-branched RNA oligonucleotides, and of the delivery to the central system of siRNA molecules comprising the linkages represented by Formula I via intrastriatal, intrathecal, lumbar intrathecal, and/or intracerebroventricular administration of Di-branched RNA oligonucleotides, are not representative or correlative of the ability to deliver adequate quantities of the claimed RNA oligonucleotides in a subject and provide therapeutic effects, as instantly claimed.
In light of the teachings in the art and the specification, one skilled in the art would not accept on its face the examples provided in the instant disclosure as being correlative or representative of the ability to provide treatment effects in a subject by any route of administration of the claimed oligonucleotides. Since the specification fails to provide the requisite guidance for the treatment in any subject, and since determination of the factors required for accomplishing this in any subject is highly unpredictable, it would require undue experimentation to practice the invention over the broad scope claimed.
For these reasons, the instant rejection for lacking enablement over the full scope claimed is proper.
Allowable Subject Matter
Claim 292 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
4-2-26
/JANE J ZARA/Primary Examiner, Art Unit 1637