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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—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
  • C12N15/1135—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 oncogenes or tumor suppressor genes
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• • A—HUMAN NECESSITIES
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

• This disclosure relates to nucleic acids and compositions thereof for inhibiting expression of the KRAS oncogene, including the KRAS mutants G12D and G12V. Also described are methods for treatment of KRAS-associated cancer, such as pancreatic cancer, using the disclosed nucleic acids.
• KRAS is a GTPase protein encoded by the Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) oncogene (National Center for Biotechnology Information (NCBI) Gene ID # 3845), which belongs to the family of RAS proteins.
• RAS proteins influence proliferation, differentiation, transformation, and apoptosis by relaying mitogenic and growth signals into the cytoplasm and the nucleolus. In a normal cell, most of the RAS molecules are present in an inactive GDP-bound conformation.
• KRAS mutations prevent dephosphorylation and inactivation of the protein, causing it to be permanently switched on, independently of EGFR-mediated signaling.
• KRAS mutations also occur in 20-30% of lung cancer patients.
• Small interfering RNA (siRNA) against mutated KRAS can lead to apoptosis of cancer cells in vitro and in vivo in mice and reduce tumor growth (Wang et al. World J. Gastroenterol. 11:2026-31 (2005); Morioka et al. In Vivo 19:535-8 (2005).; Fleming et al. Mol Cancer Res 3:413-23 (2005), Rejiba et al.
• siRNAs that have been developed, for example to silence expression of the KRAS G12V mutant (e.g., US 11,180,759 and US 10,619,159) require that the nucleotides of the siRNA be fully modified or are designed to not significantly inhibit expression of WT KRAS, which has also been implicated in cancer progression. Accordingly, a continuing need exists for compositions, such as newly developed siRNAs, for inhibiting expression of multiple KRAS mutants, while simultaneously inhibiting expression of wildtype KRAS.
• a double-stranded nucleic acid for inhibiting expression of KRAS that includes a sense strand that has a sequence of 16-25 nucleotides differing by no more than 3 nucleotides from SEQ ID NO 20 or SEQ ID NO 9; and an antisense strand that has a sequence which is substantially complementary to the sense strand sequence, wherein the antisense strand sequence includes ribonucleotides or a combination of ribonucleotides and dexoyribonucleotides, and one or more modifications selected from at least one chemically modified nucleotide, one or more substituted phosphodiester linkages between adjacent nucleotides in the sequence, a (E)-vinylphosphonate nucleotide, and one or more 3’ terminal deoxythymine nucleotides, and wherein the KRAS is wildtype KRAS, KRAS G12D, KRAS G12V, KRAS G12R, KRA
• the chemically modified nucleotide is a 2’ methoxy (2’0-Me), a deoxyribose nucleotide, or a 2’ fluoro (2’F) modified nucleotide.
• the 5’ end of the antisense strand sequence is a (E)-vinylphosphonate nucleotide.
• the one or more substituted phosphodiester linkages between adjacent nucleotides in the sequence is a phosphorothioate, thiophosphate-D-ribose, triester, thioate, or 2’ -5’ bridge covalent bond.
• the sense strand and/or the antisense strand comprises a conjugated moiety at the 5’ end, the 3’ end, or within the nucleic acid sequence, such as but not limited to a phosphatidyl serine (PS) or cholesterol conjugate or derivative thereof, a cell penetrating peptide, one or more N- Acetylgalactosamine moieties, glucagon-like peptide- 1 receptor (GLP1R), or 2'-O-hexadecyl (C16).
• PS phosphatidyl serine
• GLP1R glucagon-like peptide- 1 receptor
• C16 2'-O-hexadecyl
• the moiety is conjugated to the nucleic acid by way of a nucleotide or chemical linker.
• the sense strand comprises a cholesterol moiety conjugated to the 3’ end nucleotide with a triethyl glycol linker.
• the sense strand sequence is SEQ ID NO: 24.
• the sense strand sequence includes SEQ ID NO: 20 or 24; and the antisense strand sequence includes SEQ ID NOs 47, 51, or 56-59.
• the sense strand sequence includes SEQ ID NO: 9; and the antisense strand sequence includes SEQ ID NOs 38 or 52-55.
• the sense strand sequence includes SEQ ID NOs: 25 or 29; and the antisense strand sequence includes SEQ ID NOs 51 or 56-59.
• the sense strand sequence includes SEQ ID NO: 23; and the antisense strand sequence includes SEQ ID NOs 50, or 52- 55.
• a pharmaceutical composition comprising a double stranded nucleic acid that includes a sense strand sequence including SEQ ID NOs: 25 or 29; and an antisense strand sequence including SEQ ID NOs 51 or 56-59, and/or a double stranded nucleic acid comprising a sense strand sequence including SEQ ID NOs: 23 or 27; and an antisense strand sequence including SEQ ID NOs 50, or 52-55, and a pharmaceutically acceptable salt and/or pharmaceutically acceptable carrier.
• the described pharmaceutical composition further includes or is formulated with a biodegradable polymer, such as but not limited to PLA, PGA, PCL. PEG, PLGA, or compositions comprising combinations thereof.
• a biodegradable polymer such as but not limited to PLA, PGA, PCL. PEG, PLGA, or compositions comprising combinations thereof.
• the pharmaceutical composition is formulated as a biodegradable matrix, implant, or combination thereof.
• the pharmaceutical composition comprises multiple double- stranded nucleic acid molecules, each of which is independently formulated as a biodegradable matrix or implant, or combination thereof.
• nucleic acids and compositions described herein can be used in methods for treatment of a KRAS associated tumor including a KRAS-mutant associated tumor, by administering to a subject in need thereof, a therapeutically effective amount of the described compositions either in a monotherapy or in a combination therapy with one or more additional active agents or treatments, thereby treating the tumor.
• the methods are for treatment of a KRAS associated tumor such as but not limited to a solid tumor of a pancreatic cancer, a gastrointestinal cancer selected from the group consisting of colorectal cancer, cancer of the appendix, cancer of the esophagus, stomach cancer, and small bowel cancer; lung cancer, non-small cell non-squamous (non-Sq) NSCLC, endometrial cancer; extrahepatic cancer, or intrahepatic cholangiocarcinoma.
• a KRAS associated tumor such as but not limited to a solid tumor of a pancreatic cancer, a gastrointestinal cancer selected from the group consisting of colorectal cancer, cancer of the appendix, cancer of the esophagus, stomach cancer, and small bowel cancer
• lung cancer non-small cell non-squamous (non-Sq) NSCLC, endometrial cancer
• extrahepatic cancer or intrahepatic cholangiocarcinoma.
• Figures 1A and IB are dose response curves showing silencing activity of a combination of a G12D-targeting siRNA and a G12V-targeting siRNA, with respect to expression of a KRAS G12D expressing reporter construct ( Figure 1A) and KRAS G12V expressing reporter construct ( Figure IB).
• Figures 2A and 2B are dose response curves showing silencing activity of a combination of cholesterol-conjugated G12D-targeting siRNA and a cholesterol-conjugated G12V-targeting siRNA, with respect to expression of a KRAS G12D expressing reporter construct ( Figure 2A) and KRAS G12V expressing reporter construct ( Figure 2B).
• FIG 3 is a chart showing comparative mRNA knockdown efficacy of expression from a KRAS G12D expressing reporter construct by KRAS G12D siRNAs that are either unconjugated (“no lipid”) or conjugated to cholesterol. “Choi.” Indicates TEG- cholesterol conjugation. siRNAs were allowed to enter Panc-1 cells by free uptake, and without use of a transfection agent. siRNAs used were KRAS G12D_33 and G12D_33_Chol, as detailed in Table 1, below. [0028] Figure 4 shows a dose-response effect of KRAS mutant-targeting siRNAs on Panc-1 cellular proliferation.
• Top graph showing fold change in cell culture confluence with increasing siRNA concentration
• Bottom illustrative micrographs showing decreased conflucence of Panc-1 cells with increased siRNA concentration. As indicated, a combination of cholesterol-conjugated siRNAs targeting KRAS G12D and G12V was used.
• nucleic sequences provided herewith are shown using standard letter abbreviations for nucleotide bases as defined in 37 CFR 1.831 through 37 CFR 1.835. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
• the Sequence Listing is submitted as an XML file named 2142_19_2_seqlist.xml, created December 13, 2023, about 76.5 KB, the contents of which are incorporated by reference herein in their entirety.
• compositions means “includes.”
• consists essentially of’ or “consisting essentially of’ indicates that the active ingredient or step of the described composition or method includes only the expressly recited ingredient or step. It is to be understood that compositions that “comprise” a given ingredient can also in other embodiments “consist essentially of’ that ingredient.
• Administration The introduction of a composition into a subject by a chosen route.
• Administration of an active compound or composition can be by any route known to one of skill in the art for providing nucleic acids or nucleic acid-associated materials to a subject, including by way of a delivery device employed for or adapted for delivery of nucleic acids to a subject.
• Administration can be local or systemic. Local administration of the described compositions includes direct administration to a target of issue, such as a tumor, or into the extra-tumoral environment.
• local administration also include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intra-ocular administration, topical ophthalmic administration, or administration to the nasal mucosa or lungs by inhalational administration such as through use of an inhaler, nebulizer or similar device.
• local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ.
• local administration includes intra-arterial administration and intravenous administration when such administration is targeted to the vasculature supplying a particular organ.
• Local administration also includes the incorporation of active compounds and agents into polymeric materials which include biodegradable matrices such as micro and nanoparticles and implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects.
• biodegradable matrices such as micro and nanoparticles and implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects.
• Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system.
• systemic administration includes, but is not limited to intra-arterial and intravenous administration.
• Systemic administration also includes, but is not limited to, topical administration, subcutaneous administration, intramuscular administration, or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.
• Animal Living multi-cellular vertebrate organisms, a category that includes for example, mammals and birds.
• mammal includes both human and non-human mammals.
• subject or patient includes both human and veterinary subjects or patients, for example, humans, non-human primates, dogs, cats, horses, and cows.
• Antisense inhibitor refers to an oligomeric compound that is at least partially complementary to the region of a target nucleic acid molecule to which it hybridizes.
• an antisense inhibitor also referred to as an “antisense compound” that is “specific for” a target nucleic acid molecule is one which specifically hybridizes with and modulates expression of the target nucleic acid molecule.
• a “target” nucleic acid is a nucleic acid molecule to which an antisense compound is designed to specifically hybridize and modulate expression.
• an antisense inhibitor can be designed to target a given mutation, yet can still modulate the expression of a related sequence.
• nucleic acids described herein are designed to target the KRAS G12V or G12D mutants, but also can inhibit expression of wildtype KRAS or another KRAS G12 mutant.
• antisense inhibitory compounds can be introduced as single- stranded, double-stranded, circular, branched or hairpin compounds and can contain structural elements such as internal or terminal bulges or loops.
• Double- stranded antisense compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
• Nonlimiting examples of antisense compounds include primers, probes, antisense oligonucleotides, siRNAs, miRNAs, shRNAs and ribozymes.
• Antisense and Antisense Double- stranded DNA (dsDNA) and double- stranded RNA (dsRNA) has two strands, a 5’ -> 3’ strand, also referred to as the plus or sense strand, and a 3’ -> 5’ strand (the reverse compliment), also referred to as the minus or antisense strand.
• Antisense molecules are specifically hybridizable or specifically complementary to the sense strand of dsDNA or dsRNA.
• Sense molecules are specifically hybridizable or specifically complementary to the antisense strand of dsDNA or dsRNA.
• Neoplasia is a neoplasm (a tumor or cancer), which is an abnormal growth of tissue that results from excessive cell division.
• Neoplasia is one example of a proliferative disorder.
• a “cancer cell” is a cell that is neoplastic, for example a cell or cell line isolated from a tumor.
• Examples of solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
• pancreatic cancer that can be treated with the compositions described herein include, but are not limited to, localized pancreatic cancer such as localized pancreatic cancers such as resectable, borderline, and locally advanced tumors.
• localized pancreatic cancer such as localized pancreatic cancers such as resectable, borderline, and locally advanced tumors.
• Chemotherapeutic agent An anti-cancer agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth or hyperplasia. Such diseases include cancer, autoimmune disease as well as diseases characterized by hyperplastic growth such as psoriasis.
• diseases include cancer, autoimmune disease as well as diseases characterized by hyperplastic growth such as psoriasis.
• chemotherapeutic agent for instance, see Skeel’s Handbook of Cancer Therapy, Ninth Edition (2016) and Physicians' Cancer Chemotherapy Drug Manual 2021 21st Edition (2021).
• chemotherapeutic agents include ICL-inducing agents, such as melphalan, cyclophosphamide, cisplatin, and busulfan.
• Effective amount of a compound, therapeutically effective amount of a compound A quantity of compound sufficient to achieve a desired effect in a subject being treated.
• An effective amount of a compound can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, and particularly factors such as subject weight and metabolism, the severity and type of the affliction, and the manner of administration of the compound.
• KRAS -associated tumor Many cancerous tumors are associated with dysregulation of KRAS expression, including as a result of mutations in the KRAS gene, such as at KRAS G12. Among the most prevalent cancer types associated with KRAS are pancreatic cancers, colorectal cancers, and lung cancers. Other KRAS-associated tumors are known to the art and are encompassed by this term, including hepatobiliary tumors, breast cancer tumors, and the like. See also, Lee et al., Nature Precision Oncology, 6:91, (2022), the contents of which are incorporated by reference herein with respect to KRAS -associated tumor types.
• Injectable composition A pharmaceutically acceptable fluid composition comprising at least one active ingredient, for example, a nucleic acid, including an siRNA, a peptide, or an antibody.
• the active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, pH buffering agents and the like.
• non-toxic auxiliary substances such as emulsifying agents, preservatives, pH buffering agents and the like.
• injectable compositions that are useful for use with the compositions of this disclosure are conventional; appropriate formulations are well known in the art.
• the active ingredients can also be complexed with biodegradable polymers and processed into biodegradable matrices such as micro and/or nanoparticles.
• Neoplasia, malignancy, cancer and tumor A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.” Malignant tumors are also referred to as “cancer.”
• Nucleotide the individual molecular subunits of a polynucleotide.
• An unmodified nucleotide is one that is naturally and abundantly observed in cellular nucleic acids and composed of a nitrogenous base (Adenine - A, Guanine - G, Thymine - T, Cytosine - C, Uracil -U), a D-deoxyribose or D-ribose sugar moiety (furanose) in deoxyribonucleotides and ribonucleotides, respectively, and at least one phosphate group, which may make up an intemucleoside linkage (backbone).
• a nitrogenous base Ad-deoxyribose or D-ribose sugar moiety (furanose) in deoxyribonucleotides and ribonucleotides, respectively
• at least one phosphate group which may make up an intemucleoside linkage (backbone).
• an “unmodified nucleotide” may refer to an unmodified ribonucleotide or an unmodified deoxyribonucleotide
• a “modified nucleotide” comprises an unmodified nucleotide in which at least one of the nitrogenous base, the sugar moiety or the intemucleoside linkage is independently chemically modified.
• a modified ribonucleotide is a naturally occurring unmodified ribonucleotide, which originally comprised a D-ribose sugar moiety and a nitrogenous base selected from A, G, C and U and underwent a modification in at least one of its sugar moiety, nitrogenous base or intemucleotide linkage.
• a naturally occurring nitrogenous base in a modified ribonucleotide may be combined with a chemically modified sugar or internucleoside linkage, or vice versa, or both can be chemically modified.
• a deoxyribose nucleotide is considered to be a modified ribonucleotide.
• Uridine which is a naturally occurring ribonucleotide is considered to be a modified nucleotide in the context of DNA compounds.
• naturally occurring but non-abundant nucleotide variants such as pseudo-U or inosine (I) are also considered as modified nucleotides.
• a particular example of a modified ribonucleotide is a modification of the 2’ position of the sugar moiety, such as but not limited to addition of a methoxy group.
• a modified nucleotide is referred to as a 2'-O-Methyl sugar modified ribonucleotide.
• Other specific examples of nucleotide modifications are further described herein.
• compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed are conventional. Remington (The Science and Practice of Pharmacology, 22nd Edition (2012)), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed. In general, the nature of the carrier and/or salt will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
• non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
• pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• Pharmaceutical agent A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.
• Incubating includes exposing a target to an agent for a sufficient period of time for the agent to interact with a cell.
• Contacting includes incubating an agent in solid or in liquid form with a cell.
• Preventing or treating a disease refers to inhibiting the full development of a disease, for example inhibiting the progression or metastasis of a tumor in a subject with a neoplasm. Treatment refers to a therapeutic intervention that ameliorates or reduces a sign or symptom of a disease or pathological condition after it has begun to develop, such as the shrinkage of a tumor in a subject.
• Radiation Therapy The treatment of disease (e.g., cancer or another hyperproliferative disease or condition) by exposure of a subject or their tissue to a radioactive substance. Radiation therapy is the medical use of ionizing radiation as part of cancer treatment to control malignant cells.
• Radiotherapy may be used for curative or adjuvant cancer treatment. It is used as palliative treatment where cure is not possible and the aim is for local disease control or symptomatic relief.
• the nucleic acid molecules and compositions described herein can be used as part of a treatment regimen that includes radiation therapy.
• RNA interference A gene silencing mechanism whereby specific double stranded RNA (dsRNA) trigger the degradation of homologous mRNA (also called target RNA). Double stranded RNA is processed into small interfering RNAs (siRNA), which serve as a guide for cleavage of the homologous mRNA in the RNA induced silencing complex (RISC). The remnants of the target RNA may then also act as siRNA; thus, resulting in a cascade effect. RNAi-initiating dsRNA can also be siRNA.
• siRNA small interfering RNAs
• Small interfering RNAs Synthetic or naturally-produced small double stranded RNAs (dsRNAs) that can induce gene-specific inhibition of expression in invertebrate and vertebrate species (e.g., RNA silencing or RNAi). These RNAs are suitable for interference or inhibition of expression of a target gene and comprise double stranded RNAs of about 15 to about 40 nucleotides, which is blunt ended or contains a 3' and/or 5' overhang on each strand having a length of up to about 5-nucleotides.
• the sequence of siRNA is essentially identical to a portion of a coding region of the target gene for which interference or inhibition of expression is desired.
• the double stranded RNAs can be formed from complementary ssRNAs, from a single stranded RNA that forms a hairpin, or as the products of expression from a DNA vector.
• Substantially complementary means that most of the bases in a nucleic acid sequence can be paired with the bases in another nucleic acid strand, and mismatches or gaps are allowed (e.g., one or more nucleotides mismatches or gaps can be present).
• two “complementary” (e g., substantially complementary or completely complementary) nucleic acid sequences will selectively or specifically hybridize or anneal with each other, to form a duplex.
• Tumor bed The tissue surrounding a solid tumor, including tumor stroma.
• dsRNAs such as siRNAs
• the dsRNAs described herein include substantially complementary sense and antisense strands.
• areas of double strandedness can be found within the context of a monomeric molecule that has regions of double-strandedness resulting from internal secondary structure.
• the described dsRNAs can be used in compositions for use in treatment of KRAS -associated cancers, such as solid tumors of pancreatic cancer, colorectal cancer, and lung cancer, and include treatments for KRAS mutant-associated cancers that cannot be surgically removed.
• KRAS -associated cancers such as solid tumors of pancreatic cancer, colorectal cancer, and lung cancer, and include treatments for KRAS mutant-associated cancers that cannot be surgically removed.
• the described double stranded nucleic acid is designed to target and inhibit expression of KRAS G12D, and includes a sense strand and a substantially complementary antisense strand, each of which can be independently 16-25 nucleotides in length, wherein at least one nucleotide must be a chemically modified.
• the sense strand of the KRAS G12D double stranded nucleic acid includes a nucleotide sequence that differs in no more than three nucleotides from SEQ ID NO: 9 or SEQ ID NO: 26, while maintaining an adenosine at position 11 (counting from the 5’ end).
• the antisense strand includes a nucleotide sequence that differs no more than three nucleotides from SEQ ID NOs: 38 or 50, 52-55.
• Other sense strand sequence and antisense strand sequence combinations that can be used to target KRAS GI2D and which are encompassed by this disclosure are described in Table 1.
• the described double stranded nucleic acid is designed to target and inhibit expression of KRAS G12V, and includes a sense strand and a substantially complementary antisense strand, each of which can be independently 16-25 nucleotides in length, wherein at least one nucleotide must be a chemically modified.
• the sense strand of the KRAS G12V double stranded nucleic acid includes nucleotide sequence that differs in no more than three nucleotides from SEQ ID NOs: 20, 24, or 28, while maintaining a uracil at position 11 (counting from the 5’ end of SEQ ID NO: 20) or at position 13 (counting from the 5’ end of SEQ ID NO: 24 or 28).
• the antisense strand differs no more than three nucleotides from SEQ ID NO: 47, 51, and 56-59.
• Other sense strand sequence and antisense strand sequence combinations that can be used to target KRAS G12V and which are encompassed by this disclosure are described in Table 1.
• the double stranded nucleic acid which in particular embodiments is an siRNA
• the siRNA is 100% complementary to its target sequence.
• the siRNA is only partially complementary, and has 1, 2, or 3 nucleotides that are different from its target sequence.
• the difference from the target sequence is within the siRNA sequence itself, while in other embodiments, the difference from the target sequence is a 1, 2, or 3 base overhang at either 5’ or 3’ end, which can independently be on the sense and/or the antisense strands.
• a two-base 3' overhang is present.
• the sense strand and the antisense strand each further comprises a 2-nt 3'-overhang.
• the 3’ overhangs are made from consecutive deoxythymine (dT) nucleotides, such that a 2 nt 3’ overhang is dTdT.
• the double stranded nucleic acids described herein such as dsRNA, and more specifically siRNA, combines nucleotides that are unmodified with those that are modified, such that at least one nucleotide of the double stranded nucleic acid must be modified.
• Modifications of nucleotides can include changes to the sugar moiety, the base moiety, to the linkages between nucleotides, 1, 2, or 3 base overhangs, 5’ terminal phosphate, or a combination thereof. Modifications can also include conjugation of a non-nucleotide moiety, such as but not limited to a cholesterol moiety, at the 5’ or 3’ end.
• An unmodified (also described as a “non-modified”) nucleotide is one naturally and abundantly observed in cellular nucleic acids and composed of a nitrogenous base (Adenine - A, Guanine - G, Thymine - T, Cytosine - C, Uracil -U), a D-deoxyribose or D- ribose sugar moiety (furanose) in deoxy-ribonucleotides and ribonucleotides, respectively, and at least one phosphate group, which may make up an internucleoside linkage (backbone).
• a nitrogenous base Ad- A, Guanine - G, Thymine - T, Cytosine - C, Uracil -U
• a D-deoxyribose or D- ribose sugar moiety in deoxy-ribonucleotides and ribonucleotides, respectively
• at least one phosphate group which may make up an intern
• An unmodified nucleotide may refer to an unmodified ribonucleotide or an unmodified deoxy- ribonucleotide.
• An unmodified nucleotide in a nucleic acid molecule is covalently linked to neighboring nucleotides by phosphodiester bonds between the sugar moieties of the nucleotides via 3’-5’ links, in which the 3’ position of the nucleotide’s sugar moiety is linked through a phosphate with the 5’ position of the next nucleotide’s sugar moiety, and the 5’ position of the nucleotide’s sugar moiety is similarly linked through a phosphate with the 3’ position of the previous nucleotide’s sugar moiety (in this context, “next” and “previous” relate to the 5’>3’ direction, as commonly referred in the art).
• a modified nucleotide is an unmodified nucleotide in which at least one of the nitrogenous base, the sugar moiety, or the intemucleoside linkage is independently chemically modified.
• a modified nucleotide is a 5’ terminal nucleotide that includes modification to its 5’ phosphate group.
• deoxyribose nucleotides are considered to be modified ribonucleotides in the context of RNA molecules, such as, the described siRNA molecules.
• a nucleotide containing uracil would be considered to be a modified nucleotide in the context of DNA compounds.
• modified nucleotides include naturally occurring but non-abundant nucleotide variants, such as pseudo-U or inosine (I).
• a modified nucleotide can be one in which the sugar moiety is chemically modified.
• the modification in the sugar moiety is in the 2’-position not usually used in the native 5’ to 3 ’-intemucleoside linkage.
• modified nucleotides with a modified sugar moiety are nucleotides having carbon 2’ in the sugar moiety modified with a methyl moiety or a fluoro moiety, resulting in a 2’ O methyl (2’0-Me) modified nucleotide or a 2 ’-deoxy-2’ -fluoro (2’F) modified nucleotide, respectively.
• other positions for nucleotide modification are the 3’ and/or the 5 ’-terminal nucleotides in the sense and/or antisense strands of the dsRNA compound.
• Suitable moieties to modify the sugar moiety of a modified nucleotide include, but are not limited to: -OH; -F; -O-alkyl, -S-alkyl, or -N-alkyl; -O-alkenyl, -S-alkenyl, or -N-alkenyl; -O-alkynyl, -S-alkynyl or -N-alkynyl; or -O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl, Cl to CIO lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl (e.g. -propargyl, -propyl, -ethynyl, -
• a modified nucleotide of the described nucleic acid molecules can be a nucleotide modified in its intemucleoside linkage (also referred to as “intemucleotide linkage” or “backbone linkage”).
• a modified internucleotide linkage refers to at least one of: an internucleotide linkage other than a phosphodiester bond, an intemucleotide linkage between positions other than 3’ and 5’ within the sugar moiety of neighboring nucleotides (such as an intemucleotide linkage between position 2’ in a nucleotide’s sugar moiety and position 5’ in the next nucleotide’s sugar moiety), or a combination thereof.
• the phosphodiester bond in a modified nucleotide may be substituted with a phosphorothioate bond.
• the intemucleotide linkage in a modified nucleotide may be substituted from a 3 ’-5’ link to a 2’-5’ bridged backbone link in which the 2’ position of the sugar moiety in the modified nucleotide is linked via a phosphodiester bond to the 5’ position of the sugar moiety of the next nucleotide (in this context, “next” relates to the 5 ’>3’ direction, as commonly referred in the art), wherein such a modified nucleotide may be referred to as a 5 ’-2’ nucleotide, a 2'-5' nucleotide or a nucleotide-2’-phosphate (or a ribo-nucleotide-2’-phosphate in case of a modified ribonucleotide).
• a modified nucleotide can be nucleotide at the 5’ end of the sense or antisense strands.
• such a nucleotide is a 5’ nucleotide on the sense or antisense strand in which the terminal phosphate group has been modified, for example to be a 5’-(E)-vinylphosphonate group.
• a terminal 5’-(E)- vinylphosphonate nucleotide is a nucleotide wherein the natural phosphate group at the 5’ end has been replaced with a (E)-vinylphosphonate, which is a phosphate at the 5’ end of a nucleotide strand in which the bridging 5 ’-oxygen atom is replaced with a methynyl (-CH-) group (see International Patent Publication No. WO 2019/193144).
• the nucleic acid molecules described herein must include at least one of the foregoing modifications on the sense and/or antisense strands. Selection of the noted modifications within each strand is independent one from the other.
• one of the sense or antisense strands can be fully-modified, wherein all of the nucleotides are modified nucleotides, as a whole, the double stranded nucleic acids described herein are not fully -modified and at least one of the sense or antisense strands includes at least one unmodified nucleotide(s).
• both the sense and antisense strands are partially-modified, and include at least one unmodified nucleotide.
• the described double stranded nucleic acids can include a molecule that is conjugated at the 5’ and/or the 3’ end and/or within the sequence itself, of the sense and/or the antisense strand.
• conjugates include a lipid or other lipophilic conjugate that disrupt the cell membrane for example unsaturated fatty acids, and also including but not limited to a phosphatidyl serine (PS), cholesterol, or 2'-O-hexadecyl (C16) cholesterol. Derivatives of the foregoing conjugates could also be used.
• conjugated moieties include peptide moieties including a cell penetrating peptide, or specific cell receptor targeting peptide such as glucagon-like peptide-1 receptor (GLP1R).
• the conjugated moiety is an N-acetylgalactosamine (GalNAc) conjugate.
• the described double stranded nucleic acid is conjugated with a cell-penetrating peptide (CPP), also referred to as protein transduction domains (PTDs), which can facilitate the delivery of a molecular cargo to the cytoplasm of a cell.
• CPP cell-penetrating peptide
• PTDs protein transduction domains
• Non-limiting examples of CPP's include HIV-1 Tat (NCBI Gene ID: 155871) or a fragment thereof; pAntp (penetratin) (NCBI Gene ID: 40835); Isl-1 (NCBI Gene ID: 3670); Transportan), MPG; and Pep-1. Additional CPP's are known to those skilled in the art.
• the noted conjugate can be conjugated to the 3' end of the sense strand.
• the noted conjugate may be conjugated to the 5' end of the sense strand.
• the conjugate in the case of a hairpin- shaped molecule that includes double stranded regions, the conjugate may be conjugated to the loop.
• the conjugated moiety is bonded to a nucleotide within the described dsRNA sequence.
• the conjugated moiety is conjugated directly to the nucleotide, such as, but not limited to the 5’and/or 3’ end of the sense and/or antisense strand of the described nucleic acid.
• the conjugated moiety is separated from the 5’ and/or 3’ end by a linker, such as but not limited to a nucleotide linker (1, 2, 3, or more nucleotides) or a chemical linker, such as a C3 alkyl linker, or a triethyl glycol linker.
• a linker such as but not limited to a nucleotide linker (1, 2, 3, or more nucleotides) or a chemical linker, such as a C3 alkyl linker, or a triethyl glycol linker.
• the double stranded nucleic acid has a cholesterol moiety conjugated to the 3’ end of the sense strand by way of a tri ethyl glycol (TEG) linker.
• TAG tri ethyl glycol
• the TEG-cholesterol conjugate is of the following structure or analogous derivate thereof:
• the compounds of the present invention can be synthesized by any of the methods that are well-known in the art for synthesis of ribonucleic (or deoxyribonucleic) oligonucleotides. Such synthesis is, among others, described in Beaucage and Iyer, Tetrahedron 1992; 48:2223-2311; Beaucage and Iyer, Tetrahedron 1993; 49: 6123-6194 and Caruthers, et. al., Methods Enzymol. 1987; 154: 287-313; the synthesis of thioates is, among others, described in Eckstein, Annu. Rev.
• double stranded nucleic acid molecules described herein specifically target and inhibit (e g., “knock down” or “silence”) KRAS G12D or G12V expression.
• dsRNA compounds and siRNA compounds specifically target and inhibit (e g., “knock down” or “silence”) KRAS G12D or G12V expression.
• siRNA compounds specifically target and inhibit (e g., “knock down” or “silence”) KRAS G12D or G12V expression.
• the described compounds are designed to target KRAS G12D or G12V, it has been observed that certain embodiments of the described siRNAs which target G12D or G12V with a strong inhibitory effect, can also knock down expression of wildtype KRAS and other G12 mutants to a lesser degree.
• an siRNA described herein for targeting KRAS G12D and which strongly silences KRAS G12D expression can also silence expression, though to a lesser degree, of wildtype KRAS, KRAS G12V, KRAS G12C, KRAS G12R, KRAS G12A, and KRAS G12S.
• an siRNA described herein for targeting KRAS G12V and which strongly silences KRAS G12V expression can also silence expression, though to a lesser degree, of wildtype KRAS, KRAS G12D, KRAS G12C, KRAS G12R, KRAS G12A, and KRAS G12S.
• compositions and methods for treatment of diseases and conditions resultant from aberrant KRAS overexpression, and particularly cancers that are associated with KRAS G12 mutations can be administered to a subject in need thereof by standard modes of administration for treatment of the targeted disease, and more specifically as described below.
• the described compositions include at least one dsRNA that is designed to target KRAS G12D.
• the described compositions include at least one dsRNA that is designed to target KRAS G12V.
• the described compositions include multiple dsRNAs, at least one of which is designed to target KRAS G12D, and at least one of which is designed to target KRAS G12V.
• Particular siRNAs designed to target KRAS G12D and KRAS G12V that can be used alone or together in the described pharmaceutical compositions include, but are not limited to, a G12D-targeting siRNA having a sense strand sequence of SEQ ID NO: 23 and an antisense strand sequence of SEQ ID NO: 50 alone or together with a G12V-targeting siRNA having a sense strand sequence of SEQ ID NO: 25 and an antisense strand sequence of SEQ ID NO: 51.
• compositions include a G12D-targeting siRNA having a sense strand sequence of SEQ ID NO: 23 and an antisense strand sequence of SEQ ID NO: 54 alone or together with a G12V-targeting siRNA having a sense strand sequence of SEQ ID NO: 25 and an antisense strand sequence of SEQ ID NO: 58.
• the composition includes a G12D-targeting siRNA having a sense strand sequence of SEQ ID NO: 23 and an antisense strand sequence of SEQ ID NO: 55 alone or together with a G12V-targeting siRNA having a sense strand sequence of SEQ ID NO: 25 and an antisense strand sequence of SEQ ID NO: 59.
• KRAS G12D and/or G12V or the noted KRAS wildtype or other G12 mutant, expression by the described compositions does not require that 100% of the mRNA be silenced in the area targeted. Rather, the described methods require at least partial inhibition of KRAS G12 mutant (or wildtype) expression, such that the expression is from 0% to less than 100% of the expression that would be present in the absence of the described dsRNA compound. In particular embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or even less than 10% of unsilenced expression is necessary to achieve the desired therapeutic effect.
• the dsRNA compounds described herein, and pharmaceutical compositions thereof, can in particular embodiments be delivered to the target tissue, such as a pancreatic tumor, by direct application of the naked molecules prepared with a pharmaceutically acceptable carrier or diluent.
• naked nucleic acid or “naked dsRNA” or “naked siRNA” refers to nucleic acid molecules that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, polymeric formulations, lipofectin, or precipitating agents and the like.
• dsRNA in PBS is "naked dsRNA".
• the described dsRNA compounds, and pharmaceutically acceptable salts thereof can be delivered or administered directly with a carrier or diluent that acts to assist, promote or facilitate entry to the cell, including viral vectors, viral particles, liposome formulations, lipofectin or precipitating agents and the like.
• compositions including the described dsRNA include a pharmaceutically- acceptable delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations standard in the art.
• the dsRNA molecules disclosed herein are delivered in liposome formulations and lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
• the described RNAi agents may be complexed with a cationic molecule, such as DOTAP (N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium), DOPE (l,2-dioleoyl-sn-glycero-3 -phosphatidylethanolamine), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), spermine, PEI (polyethylenimine), or a PEI-PLA polymer.
• DOTAP N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium
• DOPE l,2-dioleoyl-sn-glycero-3 -phosphatidylethanolamine
• DOPC 1,2- di
• compositions for delivering the described nucleic acid molecules can also be composed of one or more biodegradable polymers, in which the nucleic acid, such as the dsRNA or siRNA, is incorporated into a particular polymeric matrix.
• Such polymeric compositions can be formulated into polymer- nucleic acid biodegradable matrices such as microparticles, nanoparticles, implants, or combinations thereof for sustained release delivery of the nucleic acid molecules.
• the biodegradable polymeric matrix in which the double stranded nucleic acid is incorporated is composed of poly(lactic acid) (PLA).
• the biodegradable polymeric matrix is composed of poly(glycolic acid) (PGA).
• the biodegradable polymeric matrix comprises the co-polymer of PLA and PGA known as poly(lactic-co-glycolic acid) (PLGA).
• PLA:PGA matrices of varying ratios of PLA:PGA are well known and are commercially available.
• methods for making such matrices that incorporate nucleic acid molecules such as dsRNA or siRNA are well known in the art. Exemplary methods are described in described in US Patent Application Pub. No. 2011/0195123, though it should be understood that this disclosure is not limited to only those specific polymeric matrices that have been previously disclosed.
• the PLA:PGA ratio in the PLGA copolymer is between 95:5 and 5:95, and more particularly between 25:75 and 75:25. These ratios and those following are understood to be inclusive of the noted amounts.
• the ratio is between 50:50 and 75:25, inclusive, meaning that the amount of co- polymer in the biodegradable polymeric matrix includes between 50-75% PLA and between 25-50% PGA, such as 50% PLA and 50% PGA.
• the PLA:PGA ratio is between 25:75 and 50:50, between 35:65 and 75:25, between 45:55 and 75:25, between 55:45 and 75:25, between 65:35 and 75:25, between 75:25 and 35:65, between 75:25 and 45:55, between 75:25 and 55:45, or between 75:25 and 65:25.
• the PLA:PGA ratio is between 80:20 and 90:10, inclusive.
• the PLA/PGA ratio is larger than 75:25, between 75:25 and 85:15, or between 75:25 and 95:5. Alternatively, the ratio is smaller than 25:75, between 25:75 and 15:85, or between 25:75 and 5:95.
• the co-polymer has a PLA:PGA ratio of between 80:20 and 90:10, inclusive, for example 80:20, 82:18, 84:16, 86:14, 88:12, or 90:10.
• the co-polymer has a PLA:PGA ratio larger than 75:25, for example 76:24, 78:22, 80:20, 82:18, 84:16, 86:14, 88: 12, 90: 10, 92:8, 94:6, 96:4, or 98:2.
• the co-polymer has a PLA:PGA ratio smaller than 25:75, inclusive, for example 24:76, 22:78, 20:80, 18:82, 16:84, or 14:86, 12:88, 10:90, 8:92, 6:94, 4:96, or 2:98.
• the biodegradable polymeric matrix is composed of PEG (poly(ethylene glycol), which can be the majority of the particle or implant, or used in combination with any other polymer described herein.
• polymers that can be used in the described particle or implant matrices include tri-block PLA-PCL-PLA, wherein PCL denotes poly -caprolactone; Poly(D,L-lactide) (DL-PLA), poly(D,L-glycolide); or poly(D,L-lactide-co-glycolide).
• PCL poly -caprolactone
• DL-PLA Poly(D,L-lactide)
• poly(D,L-glycolide) poly(D,L-glycolide)
• poly(D,L-lactide-co-glycolide) poly(D,L-lactide-co-glycolide).
• a polymer used in the described nucleic acid-polymeric compositions has a molecular weight (MW) of greater than 5 kilodaltons (kDa). In other embodiments, the MW is greater than 50 kDa. In other embodiments, the MW is greater than 7 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 70 kDa, 100 kDa, 150 kDa, or greater than 200 kDa. In other embodiments, the MW is between 5-100 kDa, between 7-80 kDa, 10-60 kDa, 20-50 kDa, or between 25-50 kDa.
• slow release (approximately 6 months) can be achieved, for example, with an implant containing PLGA co-polymer having a high PLA:PGA ratio, such as 90: 10, and a MW (molecular weight) higher than 50KDa.
• PLA polylactic acid
• the PLGA matrix has a PLA: PGA ratio of 65:35 and MW of 24-38 kDa. In still other embodiments, the PLGA matrix has a PLA:PGA ratio of 85:15 and MW of 190-240 kDa.
• the biodegradable matrix further comprises one or more additives for a variety of purposes including modulating hydrophilic -hydrophobic interactions; enabling dispersion of the drug, eliminating aggregation; preserving the drug in hot- temperature or cold-temperature storage conditions; and facilitating creation of cavities in the implant that affect drug diffusion from the matrix.
• Hydrophilic-hydrophobic interactions may cause aggregation of the active substance in cases of hydrophilic active substances, such as siRNA, incorporated within a hydrophobic polymer, resulting in aggregation during production or subsequently when the implant or particle formulation is administered into the body of a subject and is subjected for example to hydrolysis.
• hydrophilic active substances such as siRNA
• Non-limiting examples of such an additive to reduce such interactions are open monosaccharides, for example mannitol; disaccharides such as trehalose; sorbitol; and other cyclic monosaccharides such as glucose, fructose, galactose and disaccharides such as sucrose.
• additives when chiral, can be in the form of the D-enantiomer, the L- enantiomer, or a racemic mixture. Additional, non-limiting examples of such additives are lactose, sucrose, dextran, and hydroxyethyl starch.
• the implant or particle has between 1% and 15% mannitol, such as 1%, 1.5%, 2%, 2.5%, 5%, 7.5%, 10%, or 12.5%, and 15%, or any amount between.
• the implant or particle has less than 5% trehalose, for example in different embodiments 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5%, the effects of which on dsRNA release can be readily tested.
• the biodegradable matrix comprises an additive for protecting the dsRNA against low pH after administration in an implant or micro- or nanoparticle.
• Such a pH modulating (i.e., pH-changing) additive may be selected from bicarbonates and carbonates, for example sodium bicarbonate, sodium carbonate, and magnesium hydroxide.
• sodium bicarbonate is included at a concentration between 0.05% to about 5%, such as about 1%.
• sodium bicarbonate (or other pH modulating agent) is included at less than 1%, including 0.9%, 0.8%, 0.7%, 0.6%, 0.4% 4 and 0.2% or even less.
• sodium bicarbonate (or other pH modulating agent) is included at 2%, 3%, 4%, 5%, or any increment in between 1% and 5%.
• the described polymeric particles and implants can be coated.
• a coating can be designed for a number of characteristics, including modulating the release rate or preventing protein stickiness during long-term storage.
• the coating in some embodiments comprises the same material used to form the matrix, for example a PLGA co- polymer matrix, but without the dsRNA.
• the coating comprises a material similar to that used to form the matrix (for example containing the same building blocks in a different ratio, or containing the same polymer but with a different MW), only without the dsRNA.
• the coating comprises the same material used to form the matrix, together with at least one other polymeric material such as PEG.
• the coating includes PLA.
• the coating includes a PLGA co-polymer wherein the PLA:PGA are in a ratio of at least 80:20, for example 80:20, 82:18, 84:16, 85:15, 86: 14, 88:12, 90:10, 92:8, 94:6, 96:4, 98:2, and 99:1, and having a MW greater than that used in the polymeric matrix, such as 50KDa, for example 60KDa, 70KDa, 80KDa, 100KDa, 120KDa, 1500KDa, or 200KDa).
• the described polymer-nucleic acid matrices can be formulated into small particles, which can be administered on their own, or can be distributed within the biodegradable polymeric matrix of an implant.
• Small particles include “microparticles” and “nanoparticles.”
• Microparticles typically include particles having a size within the range 800nm - 5 pm (also referred to as microspheres).
• Nanoparticles typically include particles of size within the range 4 nm - 800 nm.
• the particles comprise a polymeric material as described herein, which can be different from or identical to that in the implant. “Different from” refers to a polymer made from different building blocks from that in the implant or even sharing at least one building block with the polymer in the implant, but having a different composition.
• the particles can be composed of PLA, whereas the surrounding matrix of the implant can be composed of PLGA.
• the differences between the polymers in the particles and the implant matrix include polymers containing a particular enantiomer as opposed to a racemic mixture of a given building block (L-PLA vs. DL-PLA), polymers containing the same building blocks in a different ratio (having either the same or different molecular weight (MW), or containing the same building blocks but having a different MW (having either the same or different ratio. “Identical to” refers to polymers with the same building blocks, in the same ratio, and with the same MW.
• particles composed of polymers that are “identical to” the constituent polymer of the implant can contain additional materials that are different from the implant matrix.
• the polymer in the particles is non-identical to the polymer in the implant.
• all of the constituent polymeric matrices are complexed with one or more of the described dsRNAs.
• a larger polymeric matrix e.g., an implant or microparticle
• the larger matrix is not complexed with the dsRNA being delivered, but the suspended smaller particles are complexed with the dsRNA being delivered.
• both the larger and smaller polymeric matrices can be complexed with a dsRNA, which in some embodiments is identical (in the multiple matrices), and in other embodiments can be different dsRNAs, including dsRNAs that target different KRAS G12 mutants.
• a dsRNA which in some embodiments is identical (in the multiple matrices), and in other embodiments can be different dsRNAs, including dsRNAs that target different KRAS G12 mutants.
• the polymeric matrix in an implant can contain a dsRNA that is designed to target KRAS G12D
• microparticles that are encompassed by the implant can contain a dsRNA that is designed to target KRAS G12V.
• the double stranded nucleic acid molecules such as dsRNA or siRNA are contained in a given biodegradable polymeric composition in a w/w nucleic acid:polymer load ratio between 1:3 and 1:100, such as above 1:100 (including 1:75, l;50, and 1:25).
• the load is above 1:20.
• the load is above 1:9.
• the load can be even above 1:3.
• the nucleic acid (e.g., RNA) release mechanism of the described RNA-polymer matrices includes in some embodiments, both bulk erosion and diffusion. In other embodiments, non-degradable or slowly degraded polymers are used, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part of the particle or implant functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months). Combinations of the different polymers described herein having different release mechanisms may also optionally be used.
• the concentration gradient at the surface can in some embodiments be constant during a significant period of the total drug releasing period, and therefore the diffusion rate is effectively constant (termed “zero mode” diffusion).
• the term “constant” refers to a diffusion rate that is maintained above the lower threshold of therapeutic effectiveness, but which may still optionally feature an initial burst and/or fluctuate, for example increasing and decreasing to a certain degree. In other embodiments, there is an initial burst of less than 10% of the total amount of drug, which may be considered negligible. In other embodiments, there is an initial burst of about 20% of the total amount of drug. In other embodiments, the design enables initial a strong burst of 30% or more of the total amount of drug.
• the diffusion rate is preferably so maintained for a prolonged period, and it can be considered constant to a certain level to optimize the therapeutically effective period, for example the effective silencing period.
• the described polymeric matrix -nucleic acid compositions can deliver its nucleic acid compound in a variable release patter, for example in which the outer portion of the polymeric matrix (e.g., the particle or implant) releases a fast loading dose, which is followed by a slow inner, osmotically released maintenance dose.
• a variable release patter for example in which the outer portion of the polymeric matrix (e.g., the particle or implant) releases a fast loading dose, which is followed by a slow inner, osmotically released maintenance dose.
• nucleic acid -polymeric matrices described herein will release dsRNA in a controlled fashion, which will vary depending on factors including but not limited to constituent polymers, additives, and surface-to-volume ratio. For example, decreasing the surface-to-volume ratio will increase the duration of dsRNA release time.
• the nucleic acid delivering biodegradable matrices such as implants, microparticles, and nanoparticles described herein are designed with a particular drug-release profile.
• One relevant parameter is the time point at which 95% of the nucleic acid has been released.
• a given polymeric matrix releases 95% of the dsRNA in vivo, for example in a human pancreatic cancer tumor or tissue surrounding the tumor, over a time period between 3-24 weeks or even months inclusive, for example 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks or months and any duration in between, for example 3-12, 2-24, 2-15, or 3-10 weeks or months inclusive.
• Another relevant parameter is the time point at which up to 90%, up to 80%, up to 70% of the active agent, up to 60%, or up to 50% has been released; this may be any of the aforementioned time frames.
• delayed-release particles or implants are utilized with the described nucleic acids.
• “Delayed-release,” as used herein, refers to polymeric matrices that do not release more than 10% of a complexed nucleic acid within the first 2 months (discounting an initial burst of up to 20%, which sometimes occurs). In other embodiments, the particles or implant do not release more than 10% of its drug load within the first 3 months. In particular embodiments, polymeric compositions containing 1% trehalose exhibit delayed release.
• the nucleic acid molecules described herein can be used in methods for treatment of a KRAS-associated cancer.
• a therapeutically effective amount of one or more of the described compositions are administered to a subject in need thereof directly into a target tumor, the cellular stroma or other tissue surrounding the tumor (synonymously described herein as the “tumor bed”), or into the blood vessels surrounding the target tumor.
• the nucleic acid molecules described herein, and pharmaceutical compositions thereof can be administered to the subject systemically, and/or by an alternate route such as parenteral or subcutaneous injection.
• KRAS associated cancers are those cancers that have been associated with aberrant KRAS overexpression.
• the aberrant KRAS overexpression results from expression of a mutant KRAS, such as a KRAS G12 mutant which includes but is not limited to KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12R, KRAS G12A, or KRAS G12S.
• a dsRNA that is designed to specifically target KRAS G12D is administered to the subject.
• a dsRNA that is designed to specifically target KRAS G12V is administered to the subject.
• multiple dsRNAs at least one of which is a dsRNA that is designed to specifically target KRAS G12D and at least one of which is a dsRNA that is designed to specifically target KRAS G12V are administered to the subject.
• KRAS-associated cancers which can be treated using the methods and compositions described herein include multiple cancers known to the art.
• KRAS associated cancers include solid tumors of pancreatic cancer, colorectal cancer, and lung cancer.
• Other particular examples of KRAS-associated cancers, and which are associated with expression of wildtype KRAS in addition to KRAS mutants such as G12D or G12V include but are not limited to gastrointestinal in general (stomach, esophagus, appendix, upper bowel, etc.) and lung cancers such as but not limited to non small cell lung cancer (NSCLC).
• NSCLC non small cell lung cancer
• the methods described herein encompass single administration of one or more KRAS G12-targeting nucleic acid(s) directly into the tumor, tumor bed, or blood vessels surrounding a target tumor, and also encompass multiple administration of one or more KRAS G12-targeting nucleic acid(s) directly into the tumor, tumor bed, or blood vessels surrounding a target tumor.
• microparticles complexed with a KRAS G12D -targeting dsRNA can be directly injected into a tumor
• microparticles complexed with a KRAS G12D-targeting dsRNA can be directly injected into several locations within a tumor, or can be injected into one or more locations within the tumor and into the surrounding tumor bed.
• nucleic acid molecules or pharmaceutical compositions disclosed herein are administered and dosed in accordance with standard medical practice, taking into account the clinical condition of the individual subject, the disease to be treated, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
• the useful dosage to be administered and the particular mode of administration will vary depending upon such factors as the cell type, or for in vivo use, the age, weight and the particular animal and region thereof to be treated, the particular nucleic acid and delivery method used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art. Typically, dosage is administered at lower levels and increased until the desired effect is achieved.
• KRAS G12-targeting nucleic acids can be administered in any one or combination of the pharmaceutical compositions described herein.
• the pharmaceutical compositions described herein for example, in complex with a biodegradable polymeric matrix, in microparticle, nanoparticle, and/or implant form.
• the described double stranded nucleic acid (e.g., dsRNA or siRNA) compositions are provided as a component of a combination therapy.
• a combination therapies provide two or more agents (e.g., two or more dsRNA or at least one dsRNA and at least another therapeutic agent) each of which is formulated and administered separately, or by administering two or more agents in a single formulation.
• agents e.g., two or more dsRNA or at least one dsRNA and at least another therapeutic agent
• Other combinations are also encompassed by combination therapy.
• two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be.
• administration of a first agent can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks.
• the two or more agents can be administered within minutes of each other or within one or several hours of each other or within one or several days of each other or within several weeks of each other. In some cases, even longer intervals are possible.
• the described combinations are provided in different formulations. In such embodiments, the different formulations can be provided by the same or different modes of administration.
• a microparticle formulation that includes a double stranded nucleic acid that targets KRAS G12D can be administered by local injection into the tumor or surrounding tumor microenvironment, whereas a separately-formulated cellular proliferation inhibitor can be provided by oral or parenteral administration.
• the two or more agents used in combination therapy may or may not be present within the patient's body at the same time.
• Combination therapy includes two or more administrations of one or more of the agents used in the combination.
• Combination treatment regimens encompassed by the methods described herein include use of the KRAS G12 mutant-targeting nucleic acid(s) as adjuvant and neoadjuvant therapies.
• the described compositions are provided with or following the main treatment for a KRAS -associated cancer (e.g., during or following a course of radiotherapy, chemotherapy, or biological therapy, and in other embodiments the described compositions are provided prior to the main treatment for a KRAS-associated cancer.
• compositions include standard of care treatments for a KRAS-associated cancer, including radiotherapy, chemotherapy (i.e., chemical), and biological (e.g., immunological (antibody, cell therapy, immune stimulatory, etc.), peptide, or other nucleic-acid based) therapies.
• radiotherapy i.e., chemical
• biological e.g., immunological (antibody, cell therapy, immune stimulatory, etc.), peptide, or other nucleic-acid based
• biological and immunological therapies are described further herein.
• Anti-cancer treatments with which the currently-described KRAS G12-mutant targeting nucleic acids can be combined are known to the art and can be determined as appropriate for treatment of a given cancer according to current knowledge and treatments (see Skeel’s Handbook of Cancer Therapy, Ninth Edition (2016) and Physicians' Cancer Chemotherapy Drug Manual 2021 21st Edition (2021)).
• KRAS mutant such as KRAS G12D and/or KRAS G12V
• KRAS G12D and/or KRAS G12V can reduce the noted immunosuppressive effects and thereby enhance the efficacy of natural CD 8+ T-cells in targeting a tumor.
• TEE tumor microenvironment
• Inhibiting expression of KRAS G12D and/or G12V, and thereby inhibiting the immunosuppresive effects of the TME is additionally associated with conversion of resident or migrating macrophages to an anti-tumor proinflammatory state.
• the described methods include additional agents that can promote or stabilize this conversion and promote macrophage phagocytosis of tumor cells.
• tumor microenvironment is synonymous with terms such as “tumor bed,” and includes the in situ tumor environment including blood vessels, immune cells, fibroblasts, signaling molecules and the extracellular matrix surrounding a solid tumor in the subject.
• Treatment of a cancer, particularly a solid tumor by administration of the described nucleic acids and compositions that inhibit KRAS G12D and/or KRAS G12V can in certain embodiments include the administration by at least one immunomodulatory agent and/or additional inhibitor of cellular proliferation. It will be appreciated that because the mechanisms of action of the various agents described herein can be complementary or even enhancing, the effect of treatments that combine the described nucleic acids with addition of an immunomodulatory agent and/or additional inhibitor of cellular proliferation can in some embodiments result in an additive effect and in some embodiments result in a synergistic effect.
• such immunomodulatory agent and/or additional inhibitor of cellular proliferation can be provided concurrently with the KRAS G12D and/or KRAS G12V inhibiting nucleic acids.
• the noted active agents can in particular embodiments be provided in the same formulation for administration to the subject. In other embodiments, the active agents are provided in different formulations.
• the immunomodulatory agent and/or additional inhibitor of cellular proliferation can be provided before administration of the described nucleic acids or compositions thereof. In yet another embodiment, the immunomodulatory agent and/or additional inhibitor of cellular proliferation can be provided after administration of the described nucleic acids or compositions thereof.
• the described active agents can additionally be provided by different routes of administration.
• the KRAS G12D and/or KRAS G12V inhibiting nucleic acids are most commonly provided locally, to the target tumor or in and around the TME.
• the described immunomodulatory agent and/or additional inhibitor of cellular proliferation can be administered locally or systemically.
• the immunomodulatory agent is an immune checkpoint inhibitory agent, such as but not limited to a checkpoint inhibitory antibody.
• Immune checkpoint inhibitory agents are capable of disrupting the signal cascade leading to T cell inhibition after T cell activation as part of what is known in the art the immune checkpoint mechanism.
• Non-limiting examples of a checkpoint inhibitory agent or checkpoint inhibitory antibody include antibodies to cytotoxic T lymphocyte antigen 4 (CTLA-4) (Uniprot Pl 6410), (programmed cell death-1) PD-1 (Uniprot Q15116), the PD-1 ligand PD-L1 (Uniprot Q9NZQ7), and B7H3 (CD276; Uniprot Q5ZPR3).
• CTLA-4 cytotoxic T lymphocyte antigen 4
• Pl 6410 Uniprot Pl 6410
• PD-1 Uniprot Q15116
• PD-1 ligand PD-L1 Uniprot Q9NZQ7
• B7H3 CD276; Uniprot Q5ZPR3
• the immunotherapy composition includes an immune checkpoint inhibitor such as a PD-1, PD-L1, and CTLA4 inhibitor.
• immune checkpoint inhibitors for use in the described methods and with the described compositions include Nivolumab (Opdivo®), a PD-1 antibody; camreliuzumab, a PD-1 antibody, spartalizumab, a PD-1 antibody, Ipilimumab (Yervoy®), a CTLA-4 antibody; Pembrolizumab (Keytruda®, MK-3475), a PD-1 antibody; atezolizumab, a PD-L1 antibody; antibodies developed by CStone Pharmaceuticals: CS1001 (anti-PD-Ll), CS1002 (anti-CTLA- 4), and CS1003 (anti-PD-1); ZKAB001 (anti-PD-Ll, China Oncology Focus/ Sorrento Therapeutics); SHR-1210 (anti-PD-1, Shanghai Hengrui
• immunotherapeutic agents are listed online at cancerresearch.org/cancer-immunotherapy/impacting-all- cancers/pancreatic-cancer, for example a PD-1 ligand (PD-Ll)bispecific fusion protein targeting PD-L1 and TGF-beta, and antibodies targeting lymphocyte activation gene 3 (LAG3), tyrosine-based inhibitory motif domain (TIGIT), and the sialic acid-binding immunoglobulin- like lectin (Siglec) family.
• PD-1 ligand PD-Ll
• LAG3 lymphocyte activation gene 3
• TGFIT tyrosine-based inhibitory motif domain
• Siglec sialic acid-binding immunoglobulin- like lectin
• dsRNAs can also be combined with immunotherapies such as mRNA vaccines, for example BNT111 (see Sahin et al., Nature 585:107-112 (2020); a whole tumor cell vaccine expressing GM-CSF (e.g., GVAX); peptide vaccines (e.g., a synthetic mutant RAS peptide); a vaccinia virus expressing the tumor antigens carcinoembryonic antigen (CEA) and/or mucin-1 (MUC-1) and the like.
• mRNA vaccines for example BNT111 (see Sahin et al., Nature 585:107-112 (2020); a whole tumor cell vaccine expressing GM-CSF (e.g., GVAX); peptide vaccines (e.g., a synthetic mutant RAS peptide); a vaccinia virus expressing the tumor antigens carcinoembryonic antigen (CEA) and/or mucin-1 (MUC-1) and the like.
• CEA
• the methods described herein include combination treatments that include providing the described double stranded nucleic acids together (e.g., in sequence or concurrently) with immunomodulatory agents that enhance the the cytotoxic capabilities of the immune system.
• immunomodulatory agents include stimulatory and co-stimulatory agents of adaptive immune cells including antigen presenting cells.
• the immunomodulatory agent is an agonist antibody targeting CD40 (see Salomon and Dahan, Front. In Immunol, doi: 10.3389/fimmu.2022.940674, 2022).
• the additional agent for combination with the described nucleic acids is a modified specificity-enhanced immune cell, such as a macrophage or T cell which has been modified ex vivo to express a chimeric antigen receptor (CAR) so as to more specifically target the tumor of interest.
• CAR-macrophages Methods of designing a CAR and their use in modifying immune cells are known in the art (see for example Sadelain et al., Cancer Disc., 3:388-398, 2013), as are their expansion and therapeutic use (Riviere et al., Mol. Ther. 25:1117-1124, 2014).
• an immunomodulatory agent can first be provided to a subject to convert a “cold” tumor into “hot” tumor that is susceptible to further treatment which is provided afterwards.
• Immune checkpoint inhibitory or blockade agents such as but not limited to antibodies targeting CTLA-4 or PD-1, have not been successful with pancreatic cancer/pancreatic ductal adenocarcinoma (PC/PDAC).
• PC/PDAC pancreatic cancer/pancreatic ductal adenocarcinoma
• Single agent blockade therapies and dual-agent blockade therapies with anti-PD-1 and anti-cytotoxic T-cell lymphocyte-4 antibodies have resulted in overall response rates of 0% and 3%, respectively (Ullman et al., J. Clin. Oncol. 40:2789, 2022).
• the anti-KRAS G12D or G1V nucleic acids described herein can result in reduced cell proliferation and an increase in cell death early in treatment.
• treatment with anti-KRAS G12D or G1V nucleic acids described herein can be combined with antiproliferative agents such as chemotherapy, radiation therapy (stereotactic or ablative), or chemo-radiation therapy that can also result in cancer antigen being released.
• antiproliferative agents such as chemotherapy, radiation therapy (stereotactic or ablative), or chemo-radiation therapy that can also result in cancer antigen being released.
• the specificity of the immune response to a tumor can be enhanced through identification of cancer neoantigens, and targeting cancer neoantigens (see Borden et al., Front. Oncol. 12:836821, 2022).
• the immunomodulatory agent is a cancer neoantigen that is provided to a tumor and either can bind to a tumor- specific cell receptor, or be expressed by the tumor, and become the target for targeted antibody therapy or an immune response against the neoantigen.
• the neoantigen is cevumeran, the overall effects of which are enhanced in combination with the nucleic acids described herein.
• the combinations of the described nucleic acids and immunomodulatory agents and/or inhibitors of cellular proliferation can in certain embodiments also include the additional provision of an inhibitor of an immune checkpoint blockade such as but not limited to an antibody targeting PD-1 or an antibody targeting CTLA-4.
• immunostimulatory cytokine Many immuno stimulatory cytokines are known to the art (see Berraondo et al., BJC, 120:6-15 (2019)), for example but not limited to interleukin- 12 (IL- 12) or variant thereof or IL-2.
• Immuno stimulatory cytokine variants that have been engineered to enhance or reduce certain properties are also included.
• the IL-2 variant bempegaldesleukin with reduced binding to CD25.
• immunomodulatory agents such as certain immunosuppressants, and inhibitors of cellular proliferation
• an siRNA(s) that targets KRAS G12D and/or G12V can be used in a treatment of pancreatic cancer along with the immunosuppressant azathioprine and/or the inhibitor of cellular proliferation MB QI 67.
• each of these agents can be separately formulated and administered to a subject in need thereof by a separate route of administration.
• nucleic acids can be provided in combination treatments with small molecule KRAS inhibitors that are known to the art, the utility of which can be extended by inclusion in such combination treatments.
• inhibitors of cellular proliferation can be used in combination with the described mutant KRAS -inhibiting nucleic acids.
• agents that activate the ERK pathway such as inhibitors of SHP2 (e.g., SHP099, TNO 155, RMC4630) and/or SOS1 (e.g., (B 13406 or BI1701963).
• the inhibitor of cellular proliferation is a nitric oxide synthase inhibitor (iNOS)/antagonist.
• iNOS nitric oxide synthase inhibitor
• iNOS for use in the current methods and combination treatments include L-citrulline, agmatine, NG, NG-dimethyl-L-arginine (ADMA), and argininosuccinic acid.
• PARP Poly (ADP-ribose) polymerase
• FAKi focal adhesion kinase inhibitors
• T regs T regulatory cells
• FAKi focal adhesion kinase inhibitors
• an FAKi can be used as a sensitizer for radiation therapy (see Osipov et al., Cancer Biol Med 2021. doi: 10.20892/j.issn.2095-3941.2020.0273).
• FAKi could sensitize the immune response.
• the described nuclei acids are provided in combination therapies with an FAKi, such as but not limited to IN10018 or VS-4718.
• Other therapies for use in combination with the described nucleic acids with and without additional combination with an FAKi include combinations with an oncolytic virus, such as an oncolytic HSV-1 virus.
• the described methods and combination therapies include combination of the described nucleic acids with an FAKi and G47D, an illustrative oncolytic HSV-1. Such combinations can be particularly useful in targeting tumors that are surrounded by a stroma-dense TME.
• inhibitors of cellular proliferation that can be used in combination with the described KRAS -inhibitory nucleic acids include antibodies or other agents targeting cellular proliferation-inducing growth factors and their cellular receptors.
• the antibody is an antibody that specifically binds to and blocks the EGFR, such as cetuximab, receptor tyrosine kinase inhibitors (e.g., famitinib, Masitinib), and tyrosine kinase inhibitors targeting angiogenesis.
• the combination therapies can include leukocyte- associated IL-like receptor 2 (LAIR2) decoy receptors, chemokine receptor 2 (CCR2) (e.g. CCX872-B, PF-04136309) and CCR5) antagonists or CCR2/CCR5 dual antagonist (e.g. BMS- 813160), inhibitors of CXCR4, and mitogen-activated protein kinase (MAPK)Zextracellular signal-regulated kinase Inhibitor (MEK) inhibitor (e.g., cobimetnib or trametinib).
• LAIR2 leukocyte- associated IL-like receptor 2
• CCR2 chemokine receptor 2
• CCR2/CCR5 dual antagonist e.g. BMS- 813160
• MEK mitogen-activated protein kinase
• MEK mitogen-activated protein kinase
• MEK mitogen-activated protein kinase
• cobimetnib or trametinib
• the inhibitor of cellular proliferation is an agent that targets KRAS protein, including mutant KRAS protein such as KRAS G12D and G12V and induces its degradation.
• KRAS protein including mutant KRAS protein such as KRAS G12D and G12V and induces its degradation.
• a particular non-limiting example of an agent that targets KRAS G12D and induces its degradation is ASP3082 (Astellas Pharma, Inc.).
• the inhibitor of cellular proliferation is an agent that binds to KRAS and reduces its activity.
• agents include molecular glues that stick to KRAS, and thereby non-covalently binds cyclophilin A to KRAS, reducing its oncological activity.
• molecular glues that can be used to this effect include RMC-6236 (Revolutions Medicine) for G12C mutations, and RMC-9805 for KRAS-G12D.
• KRAS G12D and/or G12V targeting nucleic acid can be used in treatment regimens for tumors as described.
• an immune checkpoint inhibitor can also be administered to the subject.
• certain combinations will provide even synergistic benefits over use of individual treatments.
• Example 1 Screening of dsRNAs for silencing KRAS G12D and KRAS G12V
• This example describes the generation and comparison of the silencing activity of dsRNAs designed to target KRAS G12D and KRAS G12V. All siRNA sequences for this and the following examples are listed below in Table 1.
• siRNA synthesized included sufficient material for subsequent dual dose and dose response siRNA activity screening.
• Single strand identity of the 22 siRNAs was determined by ESI-MS analysis and judged according to +/- 0.05% of calculated molecular weight.
• -Single strand purity of the synthesized oligos was sufficient if >85.0% full-length oligonucleotides (by HPLC-UV analysis: UV260nm), and duplex purity was determined as sufficient if >90.0% duplex (by SEC or non-denaturing HPLC analysis).
• Dual dose in-vitro siRNA activity screening for the 22 selected KRASG12- siRNAs was performed using the Dual Gio® co-transfection reporter assay system (Promega Corp.) according to manufacturer’s instructions. Dual Gio® mutant reporter constructs were used to assay knock down effects on KRAS wildtype, G12D, G12V, G12A, G12S, G12C, and G12R expression. Initial screening of the 22 siRNAs was done at two different siRNA concentrations (10 nM and 1 nM). All co-transfections were conducted in mouse Hepal-6 cells in quadruplicates.
• the cells were lysed 24 hours post co-transfection and the relative mRNA expression levels were quantified by determining the activity of the Renilla- and Firefly-luciferases.
• the relative target-mRNA reduction was determined by normalizing the mRNA expression levels to the expression levels in cells transfected with negative control siRNAs. A positive control siRNA was transfected to assure appropriate cell transfection efficacy.
• siRNA activity for the assayed 22 siRNAs at 10 nM concentration is shown in Table 2 and at 1 nM concentration is shown in Table 3.
• siRNAs were assayed for dose response activity, 5 siRNAs targeting KRAS G12D and 5 siRNAs targeting KRAS G12V.
• silencing activity was assayed using the Dual Gio® co-transfection reporter assay system.
• Dual Gio® mutant reporter constructs were used to assay knock down effects on KRAS wildtype, G12D, G12V, G12A, G12S, G12C, and G12R expression, but using 10 different concentrations of siRNA.
• Activity was also determined as above, with IC80, IC50, and IC20 values calculated for each siRNA tested. Results against each of the tested constructs, including maximum silencing activity are shown in Tables 4-7.
• This example shows the assay of the described cholesterol-conjugated KRAS- targeting siRNAs using the Dual Gio® co-transfection reporter assay system.
• siRNAs used were G12D_33_Chol (SEQ ID NOs: 23 and 50) and G12V_10_Chol_21 (SEQ ID NOs: 25 and 51) .
• Cells were incubated for 24 hours at 37°C/5% CO2 in a humidified incubator, followed by cell lysis and Dual Gio® assay according to manufacturer’s instructions (Promega).
• the mean ratio of R-Luc/F-Luc activity in negative control treated samples was artificially set to 100% and used for normalization of all other data points.
• Panc-1 cancer cells without use of a transfection reagent and silence KRAS expression.
• siRNAs used are listed on Table 1 as G12D_33; and G12D_33_Chol.
• Figure 3 presents the results of KRAS G12D knockdown by the indicated siRNAs.
• G12D_33_Chol (“Choi.”) provided an increased knockdown effect. This indicates that even in the absence of transfection agent, the cholesterol-conjugated siRNA can effectively enter pancreatic cancer cells and knockdown KRAS expression, and does so more effectively than the unconjugated KRAS G12D siRNA.
• siRNA efficacy was assayed in Hepa-1 cells transfected with variants of the G12D_33_ and G12V_10_21 siRNAs, as listed on Table 1, and with Dual-Glo ® reporter constructs for wildtype KRAS, KRAS G12D, and KRAS G12V as described in the preceding examples.
• siRNA efficacy was tested for both individual siRNAs, and G12D/G12V combinations. Results of these assays are shown in Tables 11-13. As shown in Tables 11-13, strong, dose dependent knockdown KRAS WT, G12D and G12V expression was observed for all tested variants, with some variations in strength of effect also observed.
• This example evaluates the efficacy of the described siRNAs formulated in polymeric microparticles in a murine S .C pancreatic tumor model.
• siRNAs are provided in several different polymeric microparticle formulations, and mixtures thereof, such as in 70%/30% ratio of one siRNA formulation to the other and vice versa.
• PANC-1 adherent cells are grown in DMEM-supplemented media. Following trypsinization, cells are washed once with DMEM-supplemented media and twice with 40 ml PBS. Cells are resuspended in PBS to 5x10 6 cells/100 ⁇ l/mouse. PANC-1 cells are injected SC (subcutaneously) into the anterior flank of each mouse. Test of siRNA efficacy is carried out after the tumor volume reaches 100 mm 3 .
• Study termination endpoints include the following: a tumor burden greater than 10% body weight; a tumor should not exceed 20 mm in any one dimension; a tumor larger than volume of 1500 mm 3 or diameter of 1.5 cm; tumors that ulcerate, become necrotic, or infected; tumors that interfere with eating or impair ambulation; palpation of tumor elicits a pain response; or mortality.
• Tumors will be also collected, photographed, weighed, placed in marked individual containers, and fixed in a 4% buffered formaldehyde solution for at least a 48-hour fixation period.
• Example 6 siRNA Stability Assessment in Biological Matrices
• This example analyzes the stability of 11 selected KRASG12-siRNAs in human serum.
• the following siRNAs will be analyzed, as detailed in Table 1: G12D_xx; G12D_33_Chol; G12V_10_Chol_21; G12D_33_Chol_varl; G12V_10_Chol_21_varl; G12D_33_Chol_var2; G12V_10_Chol_21_var2; G12D_33_Chol_var3;
• siRNA compounds are incubated for up to 48 hours in the matrix.

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