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  • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/31—Combination therapy

Definitions

• Embodiments of the present invention provide methods of stabilizing visual acuity comprising administering to a subject an effective amount of an siRNA targeting VEGF.
• the siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• Additional embodiments of the present invention provide methods of decreasing foveal thickness comprising administering to a subject an effective amount of an siRNA targeting VEGF.
• the siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• Further embodiments of the present invention provide methods of treating maeular degeneration, particularly age-related macular degeneration, comprising administering an siRNA targeting VEGF and a VEGF antagonist.
• the siRN ⁇ comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID
• Additional embodiments of the present invention provide methods of treating diabetic maeular edema comprising administering an siRNA targeting VEGF and a VEGF antagonist.
• the siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78,
• Additional embodiments of the present invention provide methods of treating diabetic retinopathy comprising administering an siRNA targeting VEGF.
• the siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• Additional embodiments of the present invention provide methods of treating proliferative diabetic retinopathy comprising administering an siRNA targeting VEGF.
• the siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• compositions comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 are provided.
• Fig, 1 is a graph of hVEGF protein levels in 293 cells trans fected with human VEGF siRNAs. non-specific siRNA (EGFP siRNA) or mock transfections without siRNA.
• Fig. 2 is a graph of the dose response studies with Cand5, hVEGF#l, hVEGF#2, hVEGF#3, hVEGF#4. hVEGF#6 and hVEGF#7.
• Fig. 3 is a graph of the mean change in best corrected distance visual acuity in human subjects as measured by the early treatment diabetic retinopathy study ("ETDRS' *" ) methodology in each of the three study groups receiving different doses of Cand5.
• EDRS' * early treatment diabetic retinopathy study
• Fig. 4 is a pair of histograms showing the percentage of human subjects in each of the study groups that exhibited stabilization of distance visua! acuity as determined by the loss of fewer than 15 letters in the ETDRS test at 12 weeks compared to baseline, or at 15 weeks as compared to 3 weeks.
• Fig. S is a graph of the mean change in best corrected near visual acuity in human subjects as measured by the ETDRS methodology in each of the three study groups receiving different doses of CandS.
• Fig. 6 is a pair of histograms showing the percentage of human subjects in each of the study groups that exhibited stabilization of near visual acuity as determined by the loss of fewer than 15 letters in the ETDRS test at 12 weeks compared to baseline, or at 15 weeks as compared to 3 weeks.
• Figs. 7A and 7B are a pair of graphs of the mean change in best corrected distance visual acuity in each of the three CandS study groups compared to the control placebo group of the related TAP study.
• Fig. 7B applies Last Observed Carried Forward (“LOCF”) analysis to the data sets.
• LOCF Last Observed Carried Forward
• Figs. 8A and SB are a pair of graphs of the change from baseline in mean size of the Choroidal Neovascularization (CNV) lesions in each of the three CandS study groups.
• Fig. 8B applies LOCF analysis to the data sets.
• Fig. 9 is a graph of the change in foveal thickness as measured by ocular coherence tomography ("OCT' * ) in three groups of human subjects with DME treated with different doses of Cand5.
• Fig. 10 is a graph of the change in visual acuity as measured by ETDRS in three groups of human subjects with DME treated with different doses of CandS .
• the term "about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
• a "subject * ' includes a human being or non-human animal.
• the subject is a human being.
• an "effective amount" of the siRNA is an amount sufficient to cause RNAi-mcd ⁇ ated degradation of the target mRNA, or an amount sufficient to inhibit the progression of angiogenesis in a subject.
• isolated means altered or removed from the natural state through human intervention.
• an siRNA naturally present in a living animal is not ''isolated,' " but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
• An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as. for example, a cell into which the siRNA has been delivered.
• target mRNA means human VEGF mRNA.
• nucleic acid sequences herein are given in the 5 1 to 3' direction. Also, all deoxytibonucl ⁇ olides in a nucleic acid sequence are represented by capital letters (e.g., deoxytlrvmidine is '"T' * ), and ribonucleotides in a nucleic acid sequence are represented by lower case letters (e.g . uridine is "u " ).
• ⁇ ngiogenesis defined as the growth of new capillary blood vessels or "neovascularization,” plays a fundamental role in growth and development. In mature humans, the ability to initiate angiogenesis is present in all tissues, but is held under strict control.
• a kev regulator of angiogenesis is vascular endothelial growth factor ("VEGF”), also called vascular permeability factor (“VPF”), VEGF exists in at least four different alternative splice forms in humans (VEGFi 2J5 VEGF 165 , VEGFs 89 and VEGF2G0X all of which exert similar biological activities.
• VEGF vascular endothelial growth factor
• VPF vascular permeability factor
• FIt-I and FIk- 1/KDR receptors also called VEGF receptor 1 and VEGF receptor 2
• FIt-I and Flk-1/KDR are transmembrane protein tyrosine kinases, and binding of VEGF initiates a cell signal cascade resulting ultimately in the neovascularization of the surrounding tissue
• Aberrant angiogenesis is implicated in a number of conditions. Among these conditions are diabetic retinopathy, diabetic macular edema ("DME”), psoriasis, exudative or '"wet" age-related macular degeneration (“ARMD”), rheumatoid arthritis and other inflammatory diseases, and most cancers.
• DME diabetic macular edema
• ARMD age-related macular degeneration
• rheumatoid arthritis and most cancers.
• the diseases associated with these conditions exhibit abnormally high levels of VEGF. and generally show a high degree of vascularization or vascular permeability.
• ARMD in particular is a clinically important angiogenic disease. This condition is characterized by choroidal neovascularization in one or both eyes in aging individuals, and is the major cause of blindness in industrialized countries,
• Diabetic macular edema also called diabetic retinopathy
• DME diabetic macular edema
• It is caused by leakiness of retinal blood vessels and the growth of new blood vessels on the retina, optic nerve and the iris.
• the leaky blood vessels result in swelling of the retina and visual loss.
• the new blood vessels that grow on the optic nerve and retina can also bleed, resulting in severe visual loss.
• new blood vessels in the iris clog the drain of the eye and can result in extremely high pressure in the eye with accompanying intense pain and the potential loss of the eye.
• DME can affect almost anyone with diabetes. In general, the longer someone has diabetes, the greater the risk of developing DME. Eventually, almost everyone with juvenile-onset diabetes will develop some symptoms of DME. Those who acquire diabetes later in life are also at risk of DME, although they are somewhat less likely to develop advanced DME.
• VEGF antagonists A number of therapeutic strategies exist for inhibiting aberrant angiogenesis, which attempt to reduce the production or effect of VEGF, otherwise referred to herein as "VEGF antagonists".
• VEGF antagonists anti-VEGF or anti-VEGF receptor antibodies, anti-VEGF aptamers, and soluble VEGF traps, which compete with endothelial cell receptors for VEGF binding have been developed.
• Classical VEGF "antisense" therapies directed against VEGF gene expression have also been proposed.
• the anti-angiogenic agents used in these therapies can produce only a stoichiometric reduction in VEGF or VEGF receptor, and the agents are typically overwhelmed by the abnormally high production of VEGF by the diseased tissue. The results achieved with available anti-angiogenic therapies have therefore been unsatisfactory.
• RNA interference is a method of post-transcriptional gene regulation that is conserved throughout many eukaryotic organisms. RNAi is induced by short (i.e., ⁇ 30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell. These short dsRNA molecules, called “short interfering RNA” or “siRNA,” cause the destruction of messenger RNAs ("mRNAs"') which share sequence homology with the siRNA to within one nucleotide resolution. It is believed that the siRNA and the targeted mRNA bind to an "RNA -induced silencing complex" or "RISC”, which cleaves the targeted mRNA.
• RISC messenger RNAs
• siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi degradation of an mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
• One embodiment of the present invention therefore provides isolated siRNA comprising short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length, that are targeted to the target mRNA.
• the siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired").
• base-paired standard Watson-Crick base-pairing interactions
• the sense strand comprises a nucleic acid sequence which is identical to a target sequence contained within the target mRNA.
• the sense and aniisense strands of the slRNA can comprise two complementary, single-stranded RNA molecules or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked, for example, by a single-stranded "hairpin" loop.
• hairpin loop of the latter type of siRNA molecule is cleaved intracellularly by the "'Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules ,
• Splice variants of human VEGF arc known, including VEGFs 2 ) (SEQ ID NO: 2).
• VEGFi6 5 SEQ ID NO: 3
• VEGFf 89 SEQ ID NO: 4
• VEGF 206 SEQ ID NO: 5
• the mRNA transcribed from the human VEGF, FIt-I SEQ ID NO: 6
• Flk-1/KDR SEQ ID NO: 7
• Such techniques include reverse transcript] on-polymerase chain reaction (RT-PCR), northern blotting and in-situ hybridization.
• databases that contain nucleotide sequences related to a given disease gene can be used to identify alternatively spliced mRNA.
• databases include GenBank, Embase, and the Cancer Genome Anatomy Project (CGAP) database.
• the CGAP database for example, contains expressed sequence tags (ESTs) from various types of human cancers.
• ESTs expressed sequence tags
• An mRNA or gene sequence from the VEGF gene can be used to query such a database to determine whether ESTs representing alternatively spliced mRNAs have been found for this gene.
• RNAse protection can also be used to identify alternatively spliced VEGF mRNA.
• RNAse protection involves transcription of a gene sequence into synthetic RNA, which is hybridized to RNA derived from other cells; for example, cells from tissue at or near the site of neovascularization. The hybridized RNA is then incubated with enzymes that recognize RNA:RNA hybrid mismatches. Smaller than expected fragments indicate the presence of alternatively spliced mRNAs.
• the putative alternatively spliced mRNAs can be cloned and sequenc ⁇ d by methods well known to those skilled in the art.
• RT-PCR can also be used to identify alternatively spliced VEGF mRNA.
• mRNA from the diseased tissue is converted into cDNA by the enzyme reverse transcriptase, using methods well-known to those of ordinary skill in the art.
• the entire coding sequence of the cDNA is then amplified via PCR using a forward primer located in the 3' untranslated region, and a reverse primer located in the 5 " untranslated region.
• the amplified products can be analyzed for alternative splice forms, for example by comparing the size of the amplified products with the size of the expected product from normally spliced mRNA, e.g., by agarose gel electrophoresis. Any change in the sue of the amplified product can indicate alternative splicing.
• mRNA produced from mutant VEGF, FIt-I or Flk-1/KDR genes can also be readily identified through the techniques described above for identifying alternative splice forms.
• "'mutant" VEGF gene or mRNA include human VEGF gene or mRNA which differ in sequence from the VEGF sequences set forth herein. Thus, allelic forms of these genes, and the mRNA produced from them, are considered “'mutants” for purposes of this invention.
• human VEGF mRNA may contain target sequences in common with their respective alternative splice forms, cognates or mutants.
• a single siRNA comprising such a common targeting sequence can therefore induce RNAi-mediated degradation of different RNA types which contain the common targeting sequence.
• the siRNA can comprise partially purified RNA. substantially pure RNA. synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
• One or both strands of the siRNA can also comprise a 3' overhang.
• a "3 * overhang” refers to at least one unpaired nucleotide extending from the 3 ' -end of a duplexed RNA strand.
• the siRNA comprises at least one 3 * overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxymicleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
• each strand of the siRNA can comprise 3 * overhangs of dithymidylic acid ("TT") or diuridylic acid ('W).
• TT dithymidylic acid
• 'W diuridylic acid
• the 3' overhangs can be also stabilized against degradation.
• the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
• substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotides in the 3' overhangs with 2 * -deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation.
• the absence of a T hydroxyl in the T- deoxythymidine significantly enhances the nuclease resistance of the 3 Overhang in tissue culture medium.
• the siRNA comprises the sequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNA comprise approximately 30-70% GiC content, and preferably comprise approximately 50% G/C content.
• the sequence of the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3 to 23), respectively, In the latter case, the 3' end of the sense siRNA is converted to TT.
• the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs.
• the antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
• the 3 '-most nucleotide residue of the antisense strand can be chosen deliberately.
• the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23-nt sense strand in either embodiment) is generally complementary to the targeted sequence.
• the siRNA comprises the sequence NAR(N 17)YNN.
• R is a purine (e.g . A or G) and Y is a pyrimidine (e.g., C or U/T).
• Y is a pyrimidine (e.g., C or U/T).
• the respective 21- nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide.
• Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol HI promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
• the siRNA comprises a sequence having no more than five (5) consecutive purines or pyrimidincs. In a further embodiment, the siRNA comprises a sequence having no more than five (5) consecutive nucleotides having the same nucJeobase (i.e., A, C, G, or U/ ' T).
• the siRNA can be targeted to any stretch of approximately 19-25 contiguous nucleotides in any of the target mRNA sequences (the '"target sequence").
• Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T et aL, "The siRNA User Guide,” the entire disclosure of which is herein incorporated by reference.
• the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.
• a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA. preferably beginning 50 to 100 nt downstream ⁇ i.e., in the 3' direction) from the start codon.
• the target sequence can, however, be located in the 5* or 3' untranslated regions, or in the region nearby the start codon (see, e.g., the target sequences of SEQ ID NOS: 73 and 74 in Table 1 below, which are within 100 nt of the 5'-end of the VEGFi 21 cDNA.
• the target mRNA sequence comprises no more than five (5) consecutive purines or pyrimidines.
• a suitable target sequence in the VEGFm cDNA sequence is:
• siRNA targeting this sequence and which has 3' uu overhangs on each strand (overhangs shown in bold), is:
• siRNA targeting this same sequence but having 3' TT overhangs on each strand (overhangs shown in bold) is:
• VEGFm target sequences from which siRNA can be derived are given in Table 1. It is understood that all VEGFi 21 target sequences listed in Table 1 are within that portion of the VEGFs 2 ! alternative splice form which is common to all human VEGF alternative splice forms. Thus, the VEGF 121 target sequences in Table 1 can also target VEGF1S5, VEGFt 89 and VEGF2 0 6 mRNA. Target sequences which target a specific VEGF isoform can also be readily identified. For example, a target sequence which targets VEGF 5 5 5 mRNA hut not VEGF m mRNA is AACGTACTTGCAGATGTGACA (SEQ ID NO: 13).
• One embodiment of the present invention provides a method of stabilizing visual acuity comprising the administration of an effective amount of an siRNA targeting VEGF.
• siRNA targeting VEGF is administered in an effective amount such that the subjects' visual acuity does not substantially decrease over time.
• siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 is administered in an effective amount such that the subjects' visual acuity does not substantially decrease over time.
• visual acuity is stabilized by administering to the subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist.
• VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumab or ranibizumab.
• the VEGF antagonist is a VEGF-TRAP, such as aflibercept.
• the VEGF antagonist is an aptamer, such as pegaptanib.
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to stabilize visual acuity is from about 0,1 mg to about 20 mg of siRNA. In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg. 2.5 mg or about 3 mg of siRNA targeting VEGF. In a preferred embodiment, the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of stabilizing the visual acuity of a subject comprising administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of stabilizing the visual acuity of a subject comprising administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every two weeks.
• a further embodiment of the invention comprises a method of stabilizing the visual acuity of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every four weeks.
• Additional preferred embodiments of the invention comprise methods of stabilizing the visual acuity of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• Another embodiment of the present invention provides a method of inhibiting choroidal neovascularization in a subject comprising the administration of an effective amount of an siRNA targeting VEGF.
• the siRNA comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 is administered to a subject in an effective amount such that the subjects' choroidal neovascularization is inhibited.
• choroidal neovascularization is inhibited by administering to a subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist.
• VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumab or ranibizumab.
• the VEGF antagonist is a VEGF-TRAP, such as aflibercept.
• the VEGF antagonist is an aptamer, such as pegaptanib
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to inhibit choroidal neovascularization is from about 0.1 mg to about 20 mg of siRNA. In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg, 2.5 mg or about 3 mg of siRNA targeting VEGF. In a preferred embodiment, the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of inhibiting choroidal neovascularization of a subject by administering an effective amount of an siRNA targeting VEGF, wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of inhibiting choroidal neovascularization of a subject by administering an effective amount of an siRNA targeting VEGF 5 wherein the effective amount of siRNA is administered every two weeks.
• a further embodiment of the invention comprises a method of inhibiting choroidal neovascularization of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every four weeks.
• Additional preferred embodiments of the invention comprise methods of inhibiting choroidal neovascularization of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• One embodiment of the present invention provides a method of treating diabetic macular edema comprising the administration of an effective amount of an siRNA targeting VEGF.
• siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 is administered to a subject in an effective amount to treat the subjects' diabetic macular edema.
• diabetic macular edema may be treated by administering to the subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist.
• VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumab or ram ' bizumab.
• the VEGF antagonist is a VEGF trap, such as aflibercept.
• the VEGF antagonist is an aptamer, such as pegaptanib.
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to treat diabetic macular edema is from about 0.1 mg to about 20 mg of siRNA. In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg, 2.5 mg or about 3 mg of siRNA targeting VEGF. In a preferred embodiment, the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of treating diabetic macular edema in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of treating diabetic macular edema in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every two weeks.
• a further embodiment of the invention comprises a method of treating diabetic macular edema of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every four weeks.
• Additional preferred embodiments of the invention comprise methods of treating diabetic macular edema of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• One embodiment of the present invention provides a method of treating diabetic retinopathy comprising the administration of an effective amount of an siRNA targeting VHGF.
• siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 is administered to a subject in an effective amount to treat the subjects' diabetic retinopathy.
• diabetic retinopathy may be treated by administering to the subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist.
• VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumab or ranibizumab.
• the VEGF antagonist is a VEGF trap, such as afiiberccpt.
• the VEGF antagonist is an aptamer, such as pegaptanib.
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to treat diabetic retinopathy is from about 0.1 mg to about 20 mg of siRNA. In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg. 2,5 mg or about 3 mg of siRNA targeting VEGF. In a preferred embodiment, the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of treating diabetic retinopathy in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of treating diabetic retinopathy in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every two weeks.
• 46- diabetic retinopathy of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• One embodiment of the present invention provides a method of treating proliferative diabetic retinopathy comprising the administration of an effective amount of an siRNA targeting VEGF.
• siRNA comprising a sense RNA slrand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 is administered to a subject in an effective amount to treat the subjects' proliferative diabetic retinopathy.
• proliferative diabetic retinopathy may be treated by administering to the subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist.
• VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumab or ranibizumab.
• the VEGF antagonist is a VEGF trap, such as aflibercept.
• the VEGF antagonist is an aptamer, such as pegaptanib.
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to treat proliferative diabetic retinopathy is from about 0.1 mg to about 20 mg of siRNA. In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg. 2.5 mg or about 3 mg of siRNA targeting VEGF, In a preferred embodiment. the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of treating proliferative diabetic retinopathy in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of treating proliferative diabetic retinopath y in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every two weeks.
• a further embodiment of the invention comprises a method of treating proliferative diabetic retinopathy of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every four weeks.
• Additional preferred embodiments of the invention comprise methods of treating proliferative diabetic retinopathy of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• One embodiment of the present invention provides a method of decreasing f ⁇ veal thickness in a subject comprising the administration of an effective amount of an siRNA targeting VEGF.
• siRNA targeting VEGF is administered to a subject in an effective amount such that the subjects' foveal thickness decreases over time.
• siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RHA strand of SEQ ID NO: 78 is administered to a subject in an effective amount such that the subjects' foveal thickness decreases.
• the foveal thickness of a subject diagnosed with diabetic macular edema is decreased by administering to the subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist.
• VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumab or ranibizumab.
• the VEGF antagonist is a VEGF trap, such as aflibercept.
• the VEGF antagonist is an aptamer, such as pegaptanib.
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to decrease foveal thickness is from about 0.1 mg to about 20 mg of siRNA. In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg. 2.5 mg or about 3 mg of siRNA targeting VEGF. In a preferred embodiment, the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an aiitisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of decreasing the foveal thickness of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of decreasing the foveal thickness of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every two weeks.
• a further embodiment of the invention comprises a method of decreasing the foveal thickness of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every four weeks.
• Additional preferred embodiments of the invention comprise methods of decreasing the foveal thickness of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• One embodiment of the present invention provides a method of treating age- related macular degeneration in a subject comprising the administration of an effective amount of an siRNA targeting VEGF.
• siRNA targeting VEGF is administered to a subject in an effective amount to treat the subjects' age- related macular degeneration
• siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an aiitisense RNA strand of SEQ ID NO: 78 is administered to a subject in an effective amount to treat the subjects' age-related macular degeneration.
• age-related macular degeneration is treated by administering to the subject an siRNA targeting VEGF and administering a pharmaceutical agent that is different from the siRNA, preferably a VEGF antagonist, VEGF antagonists comprising a number of distinct pharmaceutical classes have been developed.
• the VEGF antagonist comprises a monoclonal antibody that targets the VEGF protein, such as bevacizumah or ranibizumab.
• the VEGF antagonist is a VEGF trap, such as aflibercept.
• the VEGF antagonist is an aptamer, such as pegaptanib.
• the siRNA administered with the VEGF antagonist comprises a sense RNA strand of SEQ ID NO' 77 and an antisense RNA strand of SEQ ID NO: 78.
• the effective amount of the siRNA targeting VEGF to treat age-related macular degeneration is from about 0.3 mg to about 20 mg of siRNA, In a further embodiment, the effective amount is from about 0.2 mg to about 10 mg of siRNA targeting VEGF. In an additional embodiment, preferably the effective amount of the siRN ⁇ is from about 0.5 to about 5 mg of siRNA targeting VEGF. Further preferred embodiments provide effective amounts of about 1 mg to about 3 mg, including about 1.5 mg, 2.5 mg or about 3 mg of siRNA targeting VEGF.
• the siRNA targeting VEGF administered to a subject comprises a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78.
• An embodiment of the invention comprises a method of treating age-related macular degeneration in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered weekly.
• Another embodiment of the invention comprises a method of treating age-related macular degeneration in a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every two weeks.
• a further embodiment of the invention comprises a method of treating age-related macular degeneration of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every four weeks.
• Additional preferred embodiments of the invention comprise methods of treating age-related macular degeneration of a subject by administering an effective amount of an siRNA targeting VEGF wherein the effective amount of siRNA is administered every eight weeks, twelve weeks, sixteen weeks or twenty weeks.
• Other embodiments of the present invention provide methods of treating age- related macular degeneration and diabetic macular edema comprising administering to a subject an effective amount of a VEGF antagonist and an effective amount of an siRNA comprising a sense RNA strand and an antisense RNA strand, wherein the sense and the antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence identical to a target sequence of about 19 to about 25 contiguous nucleotides in human VEGF mRNA.
• the VEGF antagonist may be administered prior to. after or simultaneously with the siRNA.
• the sense RNA strand comprises SEQ ID NO: 77 and the antisense strand comprises SEQ ID NO: 78.
• the siRNA may be administered by intraocular administration, such as intravitreal, intraretinal, subretinal, subtenon, peri- and retro-orbital, trans-corneal and trans-scleral administration.
• the effective amount of said siRNA may be from about 0.1 mg to about 20 mg of siRNA.
• the effective amount is from about 0,2 mg to about 10 mg of siRNA targeting VEGF.
• the effective amount of the siRNA is from about 0.5 to about 5 mg of siRNA targeting VEGF.
• VEGF siRNA targeting VEGF may be administered weekly, every two weeks, every four weeks, every eight weeks, every twelve weeks, every sixteen weeks, or every twenty weeks.
• the VEGF siRNA is administered every four weeks.
• the VEGF antagonist is a monoclonal antibody targeting human VEGF, such as, for example, bevacizumab or ranibizumab.
• the VEGF antagonist is a VEGF trap, such as aflibercept.
• the VEGF antagonist is a VEGF aptamer, such as pegaptanib.
• such a method comprises administering a VEGF siRNA, preferably the sense RNA strand comprises SEQ ID NO: 77 and the antisense strand comprises SEQ ID NO: 78, and ranibizumab.
• the siRNA may be administered every four weeks (i.e. monthly) and said ranibizumab may be administered every four weeks (monthly).
• the ranibizumab may be administered two weeks prior to administration of said siRNA on an alternating basis.
• one embodiment may comprise administering an effective amount of ranibizumab on day 0, then administering an effective amount of VEGF siRNA week 2, then administering an effective amount of ranibizumab week four, then administering an effective amount of VEGF siRNA week six, then administering an effective amount of ranibizumab week eight.
• Such an administration may comprise an initiation therapy to treat the age-related macular degeneration.
• the initiation therapy may be followed by a maintenance therapy, wherein starting at week 12 the siRNA is administered, for example, every eight weeks or every twelve weeks.
• such a method comprises administering a VEGF siRNA, preferably the sense RNA strand comprises SEQ ID NO: 77 and the antisense strand comprises SEQ ID NO: 78, and bevacizumab.
• the siRNA may be administered every four weeks (i.e. monthl>) and said bevacizumab may be administered every four weeks (monthly).
• the bevacizumab may be administered two weeks prior to administration of said siRNA on an alternating basis.
• one embodiment may comprise administering an effective amount of bevacizumab on day 0, then administering an effective amount of VEGF ' siRNA week 2, then administering an effective amount of bevacizumab week four, then administering an effective amount of VEGF siRNA week six, then administering an effective amount of bevacizumab week eight.
• Such an administration may comprise an initiation therapy to treat the age-related macular degeneration.
• the initiation therapy may be followed by a maintenance therapy, wherein starting at week 12 the siRNA is administered, for example, every eight weeks or every twelve weeks,
• such a method comprises administering a VEGF siRNA, preferably the sense RNA strand comprises SEQ ID NO: 77 and the antisense strand comprises SEQ ID NO: 78, and a VEGF antagonist, such as, for example, rambizumab, bevacizumab, aflibercept or pegaptanib.
• a VEGF antagonist such as, for example, rambizumab, bevacizumab, aflibercept or pegaptanib.
• the siRNA may be administered every four weeks (i.e. monthly) and said VEGF antagonist may be administered every four weeks (monthly).
• the VEGF antagonist may be administered two weeks prior to administration of said siRNA on an alternating basis.
• one embodiment may comprise administering an effective amount of VEGF antagonist on day 0, then administering an effective amount of VEGF siRNA week 2, then administering an effective amount of VEGF antagonist week four, then administering an effective amount of VEGF siRNA week six, then administering an effective amount of VEGF antagonist week eight.
• Such an administration may comprise an initiation therapy to treat the age-related macular degeneration.
• the initiation therapy may be followed by a maintenance therapy, wherein starting at week 12 the siRNA is administered, for example, every eight weeks or every twelve weeks.
• Embodiments of the present invention also include pharmaceutical compositions comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78, wherein the siRNA is in a balanced salt solution.
• Another embodiment of the invention is a pharmaceutical composition comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 and a balanced salt solution such that the pharmaceutical composition is suitable for ocular administration.
• An additional embodiment of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 and a balanced salt solution wherein the pharmaceutical composition has a pH of about 6,08 to about 8,0.
• compositions comprising an siRN ⁇ comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 wherein the siRNA is at a concentration of 0.2 mg/100 ⁇ L to about 3.0 mg/100 ⁇ L
• additional embodiments of the invention are pharmaceutical compositions comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 wherein the siRNA is at a concentration of 1.5 mg/100 ⁇ L, 2.5 mg/100 ⁇ L or 3.0 mg/100 ⁇ L.
• compositions comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 wherein the siRNA is at a concentration of 0.2 mg/50 ⁇ L to about 3.0 mg/50 ⁇ L.
• Additional embodiments of the invention are pharmaceutical compositions comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78 wherein the siRNA is at a concentration of 1.5 mg/50 ⁇ L, 2.5 mg/50 ⁇ L or 3.0 mg/50 ⁇ L.
• Another embodiment is a pharmaceutical composition suitable for ocular administration comprising an siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78, sodium acetate (trihydrate), sodium chloride, sodium citrate(tribasic dehydrate) potassium chloride, calidum chloride (dehydrate), magnesium chloride (hexahydrate), hydrochloric acid, sodium hydroxide and water.
• siRNA comprising a sense RNA strand of SEQ ID NO: 77 and an antisense RNA strand of SEQ ID NO: 78, sodium acetate (trihydrate), sodium chloride, sodium citrate(tribasic dehydrate) potassium chloride, calidum chloride (dehydrate), magnesium chloride (hexahydrate), hydrochloric acid, sodium hydroxide and water.
• the siRNA can be obtained using a number of techniques known to those of skill in the art.
• the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the DrosophJla in vitro system described in U.S. published application 2002/0086356 of Tuschl et al.. the entire disclosure of which is herein incorporated by reference.
• the siRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
• the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
• RNA molecules or synthesis reagents Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette. CO 5 USA), Pierce Chemical (part of Perbio Science, Rockfcrd, IL, USA). G ⁇ en Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow. UK).
• the siRNA can also be synthesized as multiple complementary RNA molecules, as described in more detail in co-pending U.S. Provisional Application No. 60/824,953. entitled ⁇ siRNA and Methods of Manufacture" filed September 8. 2006, herein incorporated by reference in its entirety.
• siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
• suitable promoters for expressing siRN ⁇ from a plasmid include, for example, the U6 or Hl RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
• the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
• siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly at or near the area of neovascularization in vivo.
• the use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below.
• siRNA can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
• a plasmid comprising nucleic acid sequences for expressing an siRNA is described in Example 7 below.
• That plasmid called pAAVs ⁇ RNA, comprises a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter.
• the plasmid pAAVsiRNA is ultimately intended for use in producing a recombinant adeno-associated viral vector comprising the same nucleic acid sequences for expressing an siRNA.
• in operable connection with a polyT termination sequence means that the nucleic acid sequences encoding the sense or antisense strands are immediately adjacent to the polyT termination signal in the 5 " direction. During transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription.
• promoter under the control of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3" of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
• the siRNA can also be expressed from recombinant viral vectors intracellular ⁇ - at or near the area of neovascularization m vivo.
• the recombinant viral vectors of the invention comprise sequences encoding the siRNA and any suitable promoter for expressing the siRNA sequences. Suitable promoters include, for example, the U6 or Hl RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
• the recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver siRNA to cells in vivo is discussed in more detail below.
• siRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
• Any viral vector capable of accepting the coding sequences for the siRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associatcd virus (AAV); retroviruses (e.g, Antiviruses (LV), Rhabdoviruses, murine leukemia virus): herpes virus, and the like.
• the tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses.
• an AAV vector of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
• Preferred viral vectors are those derived from AV and ⁇ AV.
• the siRNA is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or Hl RNA promoters, or the cytomegalovirus (CMV) promoter.
• a recombinant AAV vector comprising, for example, either the U6 or Hl RNA promoters, or the cytomegalovirus (CMV) promoter.
• a suitable AV vector for expressing the siRNA, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, have been described.
• Suitable AAV vectors for expressing the siRNA, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski R ⁇ t al. (1987), J. Virol, ⁇ k 3096-3101; Fisher KJ ct al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989 ⁇ J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
• the siRNA is capable of targeting and causing the RNAi-mediated degradation of VEGF, more preferably human VEGF. Degradation of the target niRNA by the present siRNA reduces the production of a functional gene product from the VEGF,
• another embodiment of the present invention provides a method of inhibiting expression of VEGF in a subject, comprising administering an effective amount of an siRNA to the subject, such that the target mRNA is degraded.
• the products of the VEGF gene is required for initiating and maintaining angiogenesis
• another embodiment of the present invention provides a method of inhibiting angiogenesis in a subject by the RNAi- mediated degradation of the target mRNA by the present siRNA.
• RNAi-mediated degradation of the target mRNA can be detected by measuring levels of the target mRNA or protein in the cells of a subject, using standard techniques for isolating and quantifying mRNA or protein as described above.
• Inhibition of angiogenesis can be evaluated by directly measuring the progress of pathogenic or nonpathogenic angiogenesis in a subject; for example, by observing the size of a neovascularized area before and after treatment with the siRNA. An inhibition of angiogenesis is indicated if the size of the neovascularized area stays the same or is reduced.
• Techniques for observing and measuring the size of nco vascularized areas in a subject are within the skill in the art; for example, areas of choroid neovascularization can be observed by ophthalmoscopy.
• Inhibition of angiogencsis can also be inferred through observing a change or reversal in a pathogenic condition associated with the angiogenests.
• a slowing, halting or reversal of vision loss indicates an inhibition of angiogenesis in the choroid.
• f ⁇ O117j It is understood that the siRNA can degrade the target mRN ⁇ (and thus inhibit angiogenesis) in substoichiomctric amounts. Without wishing to be bound by any theory, it is believed that the siRNA causes degradation of the target mRNA in a catalytic manner.
• significantly less siRNA needs to be delivered at or near the site of neovascularization to have a therapeutic effect.
• an effective amount of siRNA is from about 0.1 mg to about 20 mg. ⁇ n other embodiments, an effective amount of siRNA is from about 0.2 mg to about 20 mg. In preferred embodiments, an effective amount of siRNA is from about 0.5 mg to about 5 mg, more preferably about 1 mg to about 3 mg, including about 1.5 mg, about 2.5 mgs and about 3.0 mg.
• angiogenic diseases include diabetic retinopathy, age-related macular degeneration (ARMD), psoriasis, rheumatoid arthritis and other inflammatory diseases. These diseases are characterized by the destruction of normal tissue by newiy formed blood vessels in the area of neovascularization. For example, in ARMD, the choroid is invaded and destroyed by capillaries. The angiogenesis-driven destruction of the choroid in ARMD eventually leads to partial or full blindness.
• age-related macular degeneration psoriasis
• rheumatoid arthritis other inflammatory diseases.
• an siRNA is used to inhibit choroidal neovascularization in age-related macular degeneration.
• the siRNA can be administered to a subject in combination with a pharmaceutical agent which is different from the present siRNA.
• the siRNA can be administered to a subject in combination with another therapeutic method designed to treat the angiogenic disease.
• the siRNA can be administered in combination with therapeutic methods currently employed for treating age- reiated macular degeneration.
• the present siRNA can be administered to the subject either as naked siRNA. in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the siRNA.
• Suitable delivery reagents for administration in conjunction with the present siRNA include the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfcctin; or polycations (e.g., polylysine), or liposomes.
• a preferred delivery reagent is a liposome.
• Liposomes can aid in the delivery of the siRNA to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the siRNA.
• Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et ai, (1980). Arm. Rev, Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4.837,028, and 5.019,369, the entire disclosures of which are herein incorporated by reference.
• the liposomes encapsulating the present siRNA comprises a ligand molecule that can target the liposome to a particular cell or tissue at or near the site of angiogenesis.
• Ligands which bind to receptors prevalent in tumor or vascular endothelial cells such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
• the liposomes encapsulating the present siRNA are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example by having opsonization- inhibition moieties bound to the surface of the structure.
• a liposome of the invention can comprise both opsonization- inhibition moieties and a ligand.
• Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
• an opsonization inhibiting moiety is '"bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a ⁇ pid- soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
• opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS' " ) and reticuloendothelial system ("RES' * ); e g,, as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference.
• Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "'stealth" liposomes.
• Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
• Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids: polyalcohols, e.g., polyvmylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GMi. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
• the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
• the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan: amirjated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
• the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
• Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes. " '
• the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinim ⁇ de ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soiublc anchor, and then bound to a membrane.
• a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive animation using Na(CN)BHj and a solvent mixture such as tctrahydrofuran and water in a 30:12 ratio at 60 0 C.
• Recombinant plasrnids which express siRNA are discussed above. Such recombinant plasmids can also be administered directly or in conjunction with a suitable delivery reagent, including the Minis Transit LTl lipophilic reagent; lipofectin; lipofcctamine; cellfectin; polycations (e.g., polylysine) or liposomes.
• a suitable delivery reagent including the Minis Transit LTl lipophilic reagent; lipofectin; lipofcctamine; cellfectin; polycations (e.g., polylysine) or liposomes.
• Recombinant viral vectors which express siRNA are also discussed above, and methods for delivering such vectors to an area of neovascularization in a patient are within the skill in the art.
• the siRNA can be administered to the subject by any means suitable for delivering the siRNA to the cells of the tissue at or near the area of neovascularization.
• the siRNA can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
• Suitable enteral administration routes include oral, rectal, or intranasal delivery.
• Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intraarterial infusion and catheter instillation into the vasculature); peri- and intra-tissue administration (e.g., peri-tumoral and intra-tumora ⁇ injection, intra-retinal injection, subretinal injection or intravitreal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct (e.g..
• a catheter or other placement device e.g., a corneal pellet or a suppository, eye-dropper, or an implant comprising a porous, non-porous, or gelatinous material
• Suitable placement devices include the ocular implants described in U.S. Pat. Nos. 5,902,598 and 6.375,972. and the biodegradable ocular implants described in U.S. Pat. No 6,331 ,313, the entire disclosures of which are herein incorporated by reference. Such ocular implants are available from Control Delivery Systems, Inc. (Watertown, MA) and Ocuiex Pharmaceuticals, Inc.
• injections or infusions of the s ⁇ RNA are given at or near the site of neovascularization. More preferably, the siRNA is administered topically to the eye, e.g. in liquid or gel form to the lower eye lid or conjunctival cul-de-sac, as is within the skill in the art (see, e.g., Acheampong AA et al, 2002, Drug Melabol. and Disposition 30: 421-429, the entire disclosure of which is herein incorporated by reference).
• the siRNA is administered topically to the eye in amounts of from about 5 microliters to about 75 microliters, for example from about 7 microliters to about 50 microliters, preferably from about 10 microliters to about 30 microliters. It is understood that topical instillation in the eye of siRNA in volumes greater than 75 microliters can result in loss of siRNA from the eye through spillage and drainage.
• a particularly preferred parenteral administration route is intraocular administration. It is understood that intraocular administration of the present siRNA can be accomplished by injection or direct (e.g., topical) administration to the eye, as long as the administration route allows the siRNA to enter the eye.
• suitable intraocular routes of administration include intravitreal, intraretinal, subretinal, subtcnon. peri- and retro-orbital, trans-corneal and trans-sclerai administration.
• intraocular administration routes are within the skill in the art; see, e.g., and Acheampong AA et ai, 2002, supra, and Bennett et al. (1996), Hum. Gene Ther.
• the siRNA is administered by intravitreal injection.
• the siRNA can be administered in a single dose or in multiple doses. Where the administration of the siRNA is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the agent directly into the tissue is at or near the site of neovascularization preferred. Multiple injections of the agent into the tissue at or near the site of neovascularization are particularly preferred.
• the siRNA can be administered to the subject once, such as by a single injection or deposition at or near the neovascularization site.
• the siRNA can be administered to a subject multiple times daily or weekly.
• the siRNA can be administered to a subject once weekly for a period of from about three to about twenty-eight weeks, more preferably from about seven to about ten weeks.
• the siRNA is injected at or near the site of neovascularization once every four weeks, eight weeks, or twelve weeks.
• the siRNA is injected at or near the site of neovascularization (e.g., intravitreally) once every four weeks during an initiation period, preferably about eight weeks, followed by a maintenance period wherein said siRNA is injected once every eight to twelve weeks.
• the dose of siRNA is preceded by administration of a VEGF antagonist, preferably two weeks prior to administration of the siRNA. and for a period of eight weeks (i.e.- the initiation period). It is understood that periodic administrations of the siRNA for an indefinite length of time may be necessary for subjects suffering from a chronic neovascularization disease, such as wet ARMD or diabetic retinopathy.
• a dosage regimen comprises multiple administrations
• the effective amount of siRNA administered to the subject can comprise the total amount of siRNA administered over the entire dosage regimen.
• the siRNA are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art.
• Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen- free.
• pharmaceutical formulations' include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
• the pharmaceutical formulations comprise an siRNA (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a physiologically acceptable carrier medium.
• Preferred physiologically acceptable carrier media are water, buffered water, saline solutions (e.g., normal saline or balanced saline solutions such as Hank's or Earle's balanced salt solutions), 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
• compositions can also comprise conventional pharmaceutical excipients and/or additives.
• Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
• Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTP A-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
• Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
• compositions of the invention for topical intraocular delivery can comprise saline solutions as described above, corneal penetration enhancers, insoluble particles, petrolatum or other gel-based ointments, polymers which undergo a viscosity increase upon instillation in the eye, or mucoadhesive polymers.
• the intraocular delivery reagent increases corneal penetration, or prolongs preocular retention of the siRNA through viscosity effects or by establishing physlcochemical interactions with the mucin layer covering the corneal epithelium.
• Suitable insoluble particles for topical intraocular delivery include the calcium phosphate particles described in U.S. Pat. No. 6,355,271 of Bell ⁇ t ah, the entire disclosure of which is herein incorporated by reference.
• Suitable polymers which undergo a viscosity increase upon instillation in the eye include p ⁇ lyethylenepolyoxypropylene block copolymers such as poloxamer 407 (e.g., at a concentration of 25%), cellulose acetophthalate (e.g., at a concentration of 30%), or a low-acetyi gellan gum such as Gelrite® (available from CP Kelco, Wilmington, DE),
• Suitable mucoadhesive polymers include hydrocolloids with multiple hydropMlic functional groups such as carboxyl, hydroxyl, amide and/or sulfate groups; for example, hydroxypropylcellulose, polyacrylic acid, high-molecular weight polyethylene glycols (e.g., >200,000 number average molecular weight),
• Suitable corneal penetration enhancers include cyclodextrins, benzalkonium chloride, polyoxyethylene glycol lauryl ether (e.g., Brij® 35), polyoxyethylene glycol stearyl ether (e.g., Brij® 78), polyoxyethylene glycol oleyl ether (e.g., Brij® 98), ethylene diamine tetraacetic acid (EDTA), digitontn, sodium taurocholate, saponins and polyoxyethylated castor oil such as Cremaphor EL.
• polyoxyethylene glycol lauryl ether e.g., Brij® 35
• polyoxyethylene glycol stearyl ether e.g., Brij® 78
• polyoxyethylene glycol oleyl ether e.g., Brij® 98
• EDTA ethylene diamine tetraacetic acid
• saponins and polyoxyethylated castor oil such as Cremaphor EL.
• solid compositions conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
• a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, preferably 25%-75%, of one or more siRNA.
• a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably l%-10% by weight, of one or more siRNA encapsulated in a liposome as described above, and propellant A carrier can also be included as desired; e.g., lecithin for intranasal delivery.
• Example 1 In Vivo RNA Interference of VEGF in Monkeys with Anti- VEGF siRNA
• siRNA duplex had the following sense and antisense strands, sense:
• siRNA sense and antisense strands formed a 19 nt double- stranded siRNA with TT 3' overhangs (shown in bold) on each strand.
• This siRNA was termed "Candidate 5" or "Cand5.”
• Other siRNA which target human VEGF mRNA were designed and tested as described for Cand5.
• CNV was induced by laser treatment to the maculae of both eyes of each animal, and the doses of CandS were given shortly following laser treatment.
• the animals were evaluated for changes in clinical signs, body weight and ocular condition (extensive ophthalmic examinations, electroretinography and tonometry). Fluorescein angiography was performed and blood samples were collected. At the end of the study (Day 44), all animals were euthanized and a complete gross necropsy was performed. Selected tissues were collected and preserved for histopathologic evaluation.
• Example 2 In Vitro RNA Interference of VEGF with Anti-VEGF siRNA in Human Embryonic Kidney 293 Celts
• Human embryonic kidney 293 cells obtained from ATCC, Manassas, VA were cultured in Dulbecco's Modified Eagle Medium (DMEM; obtained from Cellgro, Herndon, VA) with 10% fetal bovine serum (FBS; from JRH Biosciences, Lenexa. KS) and an antibiotic-antimycotic reagent, used for the prevention of cell culture growth contaminants (from Gibco, Carlsbad, CA).
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• an antibiotic-antimycotic reagent used for the prevention of cell culture growth contaminants (from Gibco, Carlsbad, CA).
• siRNAs were synthesized by Integrated DNA Technologies (Coralville, IA). The siRNA target sequences are shown in Table 2. An additional siRNA was used in this study that targets the gene of enhanced green fluorescent protein (EGFP) as a negative control.
• EGFP enhanced green fluorescent protein
• siRNA Transfeciion and Hypoxia Induction In Vitro. Human 293 cells were cultured in 24 well plates at 37 0 C with 5% CO2 overnight. The next day, transfections were performed when cells were about 50%-70% confluent. Cells were transfected with siRNAs directed against human VEGF. siRNAs were mixed in a CaPi reagent and added to 20 ⁇ l of 250 rnM CaC ⁇ 2 solution. The siRNA/CaCh mixture was added drop-wise to 20 ⁇ l of 2X Hanks Balanced Salt Solution (HBS), while mixing by vortex. The siRN ⁇ /CaCh/HBS complex was added directly to the medium in each well (300 ⁇ L/well).
• HBS Hanks Balanced Salt Solution
• DMSO-containing serum-free medium 300 ⁇ L/well at room temperature for 1-2 minutes. This medium was then removed, and the cells were fed again with growth medium (500 ⁇ L/well).
• siRNAs were used at a concentration of 25nM.
• siRNAs were used at concentrations of 1 nM. 5nM and 25nM.
• Hypoxia was induced with desferoxamine at a final concentration of 130 uM 4 hours after transfection was performed. Desferrioxamine mimics a hypoxic state, as it is proposed to disrupt normal oxygen-sensing pathways in mammalian cells by inhibiting heme- Fe2+ interactions.
• VEGF Protein Quantification Approximately 48 hours post transfection, the supernatant was removed from all wells and a human VEGF ELISA (R & D systems, Minneapolis, MN) was performed on the 293 cells as described in the Quantikine human VEGF ELISA protocol. VEGF-specific Antibody was added to each well causing color development in proportion to the amount of VEGF bound to the plate. ELISA results were read on an AD340 plate reader at 450 nm (Beckman Coulter).
• Human VEGF siRNAs Suppresses Hypoxia-Induced Up-regulation of Human VEGF Protein in 293 Cells. Human VEGF was upregulated by the desferrioxamine-mediated induction of hypoxia. Readings of OD 450nm reflected the human VEGF protein levels in cell samples. The hypoxia-induced increase of hVEGF protein levels were significantly reduced in cells transfected with all of the human VEGF siRNAs ( Figure 1). No effect on hVEGF levels were observed with transfections with nonspecific siRNA (EGFP siRNA) or mock transfections without siRNA.
• EGFP siRNA nonspecific siRNA
• CNV subfoveal choroidal neovascularization
• Minimally classic lesions were to have evidence oi * lesion activity defined as: a. The presence of subretinal hemorrhage and/or fluid and/or lipid OR b. Loss of one or more lines of vision (ETDRS or equivalent; Early Treatment Diabetic Retinopathy Study visual acuity charts) during the past 12 weeks
• ETDRS best corrected visual acuity of 64 to 24 letters (20/50 to 20/320 Snellen Equivalent) in the study eye;
• Subretinal hemorrhage no more than 50% of total lesion size, and may not involve the subfoveal space;
• the last observed efficacy values prior to the rescue therapy were Io be used as the LOCF value.
• the analyses on the available observed data without imputation was performed. Unless otherwise specified, analyses is on the available observed data.
• BCDVA Best Corrected Distance Visual Acuity
• Table 4 Summary of BCDVA increase from baseline - Subjects gaining 15, 10, 5 or any letters.
• Table 5 Summary of BCDVA increase from week three - Sub ects gaining 15, 10, 5 or aay letters.
• N ⁇ number of subjects with available daia n - number of subjects w ith the corresponding VA response in each dose group
• Stabilization of best corrected acuity defined as no loss of 15 letters and considered the primary analysis in clinical trials of therapeutic agents for CNV, is shown in Tables 6 and 7, and Fig. 4. As shown in Table 6, approximately 85% of eyes remained stable through 9 weeks; this decreased somewhat to 78% at 12 weeks, and then remained relatively stable at 77% and 83% at 15 and 18 weeks, respectively. Table 6: Summary of BCDVA decrease from baseline - Subjects not losing IS, 10, 5 or any letters.
• N number of subjects with available data.
• n number of subjects with the corresponding VA response in each dose group.
• Table 7 Summary of BCDVA decrease from week three - Subjects not losing 15, 10, 5 or any letters.
• N number of subjects with available data.
• n number of subjects with the corresponding VA response in each dose group.
• BCNVA near visual acuity
• Stabilization of best corrected near acuity i.e., the proportion of subjects not losing 15 letters, and loss of letters of BCNVA are summarized in Table 9 and Fig. 6. Al week 9, over 90% of the study population had not lost 15 letters of near acuity, and only a small decrease in this proportion of responders was observed at 12, 15 and 18 weeks. Stability of the best corrected near acuity was even more notable when considering the change from 3 weeks to later visits (Fig. 6).
• Table 9 Summary of BCNVA decrease from baseline Subjects not losing 15, 10, 5 or any letters.
• N number of subjects with available data
• n number of subjects with the corresponding VA response in each dose group % - n ⁇ M ⁇ 100%
• VEGF Choroidal Neovascularization
• Fig. 8A and 8B LOCF
• VEGF has been shown to be both necessary and sufficient for the growth of CNV in animal models (Adamis AP et al. (1996) Arch Ophthalmol 114:66-71), and therefore is expected to be the most VEGF responsive component of the lesion. It is noteworthy to point out that there is evidence of a dose effect trend, that while not statistically significant is apparent at all visits in the growth of CNV from baseline.
• Table 10 Fluoroscein Angiography - total lesion size.
• Baseline the most recent non-missing data prior to the treatment injection at Day 0.
• N number of subjects with data.
• CNV Percent of Total Lesion Size The change in the proportion of the total lesion represented by CNV is shown in Table 11. As shown, there was an overall decrease in the CNV percent of total lesion size in all three treatment groups, with increasing magnitude of this change from week 3 through week 18. Table 11: FIuoroseein Angiography change from baseline - choroidal neovascularization % of lesion.
• N number of subjects with available data.
• CandS may act by silencing VEGF expression., but is not a VEGF antagonist and has no action on previously produced VEGF protein.
• the increased circulating levels of VEGF in various tissues of the eye continue to promote neovascularization and vascular permeability in the absence of new VEGF production until the protein is turned over by receptor binding and/or natural catabolic processes.
• the efficacy of CandS in inhibiting VEGF production may not be immediately manifest until this "turnover" of protein occurs. As this extracellular VEGF protein population turns over, new VEGF production would be inhibited by CandS.
• the analysis of change from 3 weeks to 12, 15 and 18 weeks for each key outcome may represent a more pharmacologically relevant time window for observing CandS efficacy.
• This analysis assumes turnover and degradation of extracellular VEGF present in the retinal tissues in approximately 3 weeks, although this has not been definitively established.
• CNV size changed only minimally in this 18 week period, suggesting stabilization of the lesion.
• VEGF has been shown to be both necessary and sufficient for the growth of CNV in animal models [3], and therefore CNV is expected to be the most VEGF responsive component of the lesion.
• the baseline age was 62.7 years, 56% female and 44% male, 70% Caucasian and 30% non-Caucasian. In the fellow eye, 76.7% had DME and 23.3% did not have DME.
• the mean visual acuity was 57.3 letters (20/63).
• Using the international clinical diabetic retinopathy disease severity scale 6 were mild non-proliferative, 18 were moderate nonproliferative, 8 were severe non-proliferative and 16 were proliferative.

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