Short hairpin RNA-mediated inhibition of HERV-k expression and uses thereof

Abstract

Short hairpin RNA (shRNA) molecules that target human endogenous retrovirus K (HERV-K) nucleic acid, as well as vectors containing the coding sequences for the shRNA molecules, are described. In some instances, the vector containing the shRNA coding sequences is an adeno-associated virus (AAV) vector, such as a self-complementary AAV vector. Use of the shRNA molecules, vectors and compositions thereof for the treatment of neurological disorders, such as motor neuron diseases, and cancer is also described.

Description

[0001] 4239-1 10530-02

[0002] SHORT HAIRPIN RNA-MEDIATED INHIBITION OF HERV-K EXPRESSION AND

[0003] USES THEREOF

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 680,228, filed August 7, 2024, which is herein incorporated by reference in its entirety.

[0006] FIELD

[0007] This disclosure concerns short hairpin RNA (shRNA) molecules that target the human endogenous retrovirus K (HERV-K) envelope (Env) gene and adeno-associated virus (AAV) vectors containing the HERV-K-targeted shRNAs. Use of the shRNAs and AAV vectors for the treatment of neurological conditions and tumors is also described.

[0008] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0009] The electronic sequence listing, submitted herewith as an XML file named 4239- 110530-02.xml (43,822 bytes), created on July 24, 2025, is herein incorporated by reference in its entirety.

[0010] BACKGROUND

[0011] Human endogenous retroviruses (HERVs) are ancient viruses that integrated into the human germline millions of years ago and now comprise approximately 8% of the human genome. However, they have acquired mutations that have rendered them replication incompetent. HERVs have been associated with the pathogenesis of some cancers as well as neurological diseases. Human endogenous retrovirus K (HERV-K) subtype HML-2 has the ability to produce a functional envelope protein and virus-like particles from multiple loci within the human chromosome. A subset of patients with amyotrophic lateral sclerosis (ALS) have increased expression of HML-2 and transgenic mice with the HML-2 envelope develop an ALS -like phenotype. Thus HML-2 is a potential target for the treatment of ALS and other neurological disorders.

[0012] SUMMARY

[0013] Described herein are short hairpin RNA (shRNA) molecules that block or inhibit expression of HERV-K Env, Gag and Pol genes and recombinant viral vectors encoding the disclosed HERV-K-targeted shRNA molecules. The shRNA molecules and vectors can be 4239-1 10530-02 used to inhibit expression of HERV-K genes in vivo. In view of the association between HERV-K and neurological diseases and cancer, the disclosed shRNA molecules and vectors can also be used for treating such diseases.

[0014] Provided herein are isolated shRNA molecules targeting a HERV-K nucleic acid molecule, wherein the shRNA molecule is encoded by a nucleic acid sequence including or consisting of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

[0015] Also provided are vectors, such as viral vectors (<?.g., an AAV vector) that include a coding sequence for an shRNA molecule targeting a HERV-K, wherein the coding sequence for the shRNA molecule includes or consists of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. In some aspects, the vector includes coding sequences for multiple different shRNA molecules, such as two or three shRNA molecules. In specific examples, the vector includes coding sequences for three different shRNA molecules and the coding sequences include or consist of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5. In some examples, the vector is a self-complementary AAV vector.

[0016] Isolated cells that include an shRNA molecule or a vector disclosed herein are also provided. In some aspects, the cell is a mammalian cell.

[0017] Further provided are compositions that include a pharmaceutically acceptable carrier and at least one isolated shRNA molecule or vector disclosed herein.

[0018] Also provided is a method of inhibiting expression of HERV-K gene expression in a subject. In some aspects, the method includes administering to the subject an effective amount of an shRNA molecule, vector, or composition disclosed herein.

[0019] Further provided is a method of treating a neurological disorder in a subject by administering to the subject a therapeutically effective amount of an shRNA molecule, vector, or composition disclosed herein. In some aspects, the neurological disorder is a motor neuron disease, Alzheimer's disease, schizophrenia, or frontotemporal dementia. In some examples, the motor neuron disease is amyotrophic lateral sclerosis (ALS), spinal muscular 4239-1 10530-02 atrophy (SMA), spinal muscular atrophy with respiratory distress type 1 (SMARD1), congenital SMA with arthrogryposis, progressive bulbar palsy (PBP), primary lateral sclerosis, progressive muscular atrophy (PMA), Kennedy’s disease, or post-polio syndrome (PPS).

[0020] Also provided are methods of treating cancer in a subject by administering to the subject a therapeutically effective amount of an shRNA molecule, vector, or composition disclosed herein. In some aspects, the cancer is a brain tumor (e.g., atypical teratoid rhabdoid tumor, medulloblastoma, glioblastoma, or glioma), breast cancer, prostate cancer, melanoma, a germ cell tumor, ovarian cancer, pancreatic cancer, or hepatocellular carcinoma.

[0021] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGS. 1A-1C: Inhibition of HERV-K Env expression by single shRNAs targeted to HERV-K Env. HEK293 cells were co-transfected with a HERV-K plasmid and an adeno- associated virus (AAV) vector containing a single shRNA targeted to the HERV-K Env sequence. Fourteen shRNAs were tested (see Table 1; SEQ ID NOs: 1-14) and compared to a previously described HERV-K- targeted shRNA (referred to as shRNA 1000). Transfected cells were harvested for qPCR determination of Env mRNA at 24 hours (FIG. 1 A), 48 hours (FIG. IB), and 72 hours (FIG. 1C) after transfection. The inhibition of Env expression by each shRNA is shown as fold change compared to a control shRNA (LacZ shRNA).

[0024] FIG. 2: Inhibition of HERV-K Env, Gag and Pol expression by single shRNAs targeting HERV-K Env. HEK293 cells were co-transfected with a HERV-K plasmid and an AAV vector containing a single shRNA. Eight shRNAs were tested (231, 235, 238, 286, 343, 470, 878, and 1033), along with shRNA 1000. Transfected cells were harvested for qPCR determination of Env, Gag, and Pol mRNA at 48 hours after transfection. The inhibition of Env, Gag or Pol expression by each shRNA is shown as fold change compared to a control shRNA (LacZ shRNA).

[0025] FIG. 3: Inhibition of HERV-K expression in HEK293 cells co-transfected with full- length HERV-K and HERV-K Env tandem shRNA. HEK293 cells were co-transfected with a HERV-K full length plasmid and an AAV vector with either a single shRNA (235, 286 or 343) or a combination of two (235-286) or three (235-286-343) shRNAs in tandem. At 4239-1 10530-02 different time points after transfection (24, 48, and 72 hours), cells were harvested and subjected to qRT-PCR for HERV-K viral gene mRNA (Env, Gag, and Pol). A random shRNA without a human gene target was used as a control. GAPDH was used as an internal mRNA standard. The level of mRNA is presented as fold change compared to the control. The tandem shRNA 235-286-343 construct was the most effective at inhibiting HERV-K viral gene expression.

[0026] FIGS. 4A-4C: HERV-K Env shRNA blocks Env protein expression. HEK293 cells were co-transfected with a HERV-K full length plasmid and an AAV vector with either a single shRNA (235, 286 or 343) or a combination of two (235-286) or three (235-286-343) shRNAs in tandem. At different time points after transfection (24, 48, and 72 hours), cells were harvested and subjected to Western blot analysis to detect Env protein expression. A random shRNA without a human gene target was used as a control. Vinculin was used as an internal protein expression control. The tandem shRNA 235-286-343 was most effective at inhibiting Env protein expression.

[0027] FIG. 5: HERV-K Env shRNA blocks viral gene mRNA from different chromosomal loci. HEK293 cells were co-transfected with a full length HER V -K plasmid from one of several different chromosomal loci (5q33.3, 6ql4.1, 7p22.1, 12ql4.1 , or 19ql2b) and an AAV vector with tandem shRNA (shRNA 235-286-343). At 72 hours after transfection, cells were harvested and subjected to qRT-PCR analysis for HERV-K viral gene mRNA (Env, Gag, and Pol). A random shRNA without a human gene target was used as a control. GAPDH was used as an internal mRNA standard. The level of mRNA is presented as fold change relative to the control. The tandem shRNA blocked HERV-K viral gene expression from all tested loci.

[0028] FIG. 6: Plasmid map of the tandem shRNA vector pAAV-U6-shRNA235-shRNA286- shRNA343, referred to herein as sHA-9.

[0029] FIG. 7: Plasmid map of the self-complementary AAV vector pscAAV-U6- shRNA235-shRNA286-shRNA343. Three shRNAs (shRNA235, shRNA286, and shRNA343) directed against human endogenous retrovirus HERV-K subtype HML-2 were inserted in tandem as self-complementary DNA in a plasmid with the inverted terminal repeat (ITR) from adeno-associated virus-2 (AAV-2). Once pseudotyped with AAV-9, the vector is termed “sHscA-9.”

[0030] FIG. 8: Effect of sHscA-9 on HML-2 transcripts. Tera-1 cells were seeded in 12-well plates at a density of 1x10scells per well and infected with sHscA-9 (“scAAV”) or sHA-9 (“ssAAV”) in serum free media. An AAV-9 plasmid without the shRNAs was used as a 4239-1 10530-02 control (control AAV). RNA was extracted from the cells at 72 hours and analyzed by RT- PCR for each of the HERV-K subtype HML-2 transcripts. The decrease in transcripts observed using scAAV was more robust compared to ssAAV.

[0031] SEQUENCES

[0032] The nucleic acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases as defined in 37 C.F.R. 1 .822. 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. In the accompanying sequence listing:

[0033] SEQ ID NOs: 1-24 are nucleic acid sequences encoding shRNA molecules targeting HERV-K Env.

[0034] SEQ ID NOs: 25-48 are HERV-K Env gene sequences targeted by the disclosed shRNA molecules.

[0035] DETAILED DESCRIPTION

[0036] I. Abbreviations

[0037] AAV adeno-associated virus vector

[0038] ALS amyotrophic lateral sclerosis

[0039] AT / RT atypical teratoid rhabdoid tumor

[0040] Env envelope

[0041] GBM glioblastoma multiforme

[0042] HERV human endogenous retrovirus

[0043] ITR inverted terminal repeat

[0044] MMN multifocal motor neuropathy

[0045] PBP progressive bulbar palsy

[0046] PLS primary lateral sclerosis

[0047] PMA progressive muscular atrophy

[0048] Pol polymerase

[0049] PPS post-polio syndrome qPCR quantitative polymerase chain reaction scAAV self-complementary AAV shRNA short hairpin RNA

[0050] SMA spinal muscular atrophy 4239-1 10530-02

[0051] SMARD1 spinal muscular atrophy with respiratory distress type 1 ssAAV single- stranded AAV ssDNA single- stranded DNA

[0052] IL Summary of Terms

[0053] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an shRNA” includes singular or plural shRNAs and can be considered equivalent to the phrase “at least one shRNA.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0054] Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for therapeutic use. There are currently 11 recognized serotypes of AAV (AAV 1-11). In some aspects herein, the AAV is AAV9, or AAV2 pseudotyped with AAV9. A pseudotyped AAV vector refers to an AAV vector in which the viral capsid proteins are derived from a first AAV serotype and the viral DNA is from a second AAV serotype. In some aspects herein, the AAV vector is a self-complementary AAV (scAAV) vector. Self- complementary AAV vectors have a double-stranded genome, whereas traditional AAV vectors have a single- stranded genome. scAAV vectors typically exhibit more rapid and efficient gene expression because they already contain a double-stranded DNA structure, eliminating the need for second-strand synthesis. scAAV vectors have a modified ITR that 4239-1 10530-02 allows for packaging of both the sense and antisense strands of the DNA into a single vector particle.

[0055] Administration / Administer: To provide or give a subject an agent, such as a therapeutic agent (e.g. a shRNA molecule or a recombinant AAV encoding a shRNA), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intratumoral, or renal vein injection), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0056] Alzheimer's disease: A chronic neurodegenerative disease that is the cause of 60% to 70% of cases of dementia. The most common early symptom is difficulty in remembering recent events (short-term memory loss). As the disease advances, symptoms can include problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, not managing self-care, and behavioral issues. Although the speed of progression can vary, the average life expectancy following diagnosis is three to nine years. Alzheimer’s disease is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions of the brain. This loss results in gross atrophy of the affected regions, including degeneration in the temporal lobe and parietal lobe, and parts of the frontal cortex and cingulate gyrus. Degeneration is also present in brainstem nuclei like the locus coeruleus. Both amyloid plaques and neurofibrillary tangles are clearly visible by microscopy in brains of patients. The plaques are dense, mostly insoluble deposits of betaamyloid peptide and cellular material outside and around neurons. Tangles (neurofibrillary tangles) are aggregates of the microtubule-associated protein Tau that accumulate inside the cells themselves.

[0057] Amyotrophic lateral sclerosis (ALS): A motor neuron disease, formerly known as Lou Gehrig's disease, that can affect the upper and / or lower motor neurons. ALS causes rapid loss of muscle control and eventual paralysis. ALS may also be associated with frontotemporal dementia.

[0058] Atypical teratoid rhabdoid tumor (AT / RT): A rare and aggressive central nervous system tumor that begins in the brain or spinal cord. AT / RT usually occurs in infancy and early childhood.

[0059] Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma and mucinous carcinoma. Triple negative 4239-1 10530-02 breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2 / neu protein. Triple negative breast cancer is also called ER-negative PR-negative HER2 / neu-negative breast cancer.

[0060] Congenital SMA with arthrogryposis: A rare motor neuron disease that appears at birth. Symptoms include severe joint contractures, making babies unable to extend or flex the affected joints. In most children, both the arms and legs are affected. Other symptoms include scoliosis (curvature of the spine), chest deformity, respiratory problems, unusually small jaw, and drooping eyelids.

[0061] Frontotemporal dementia: A group of brain diseases that primarily affect the frontal and temporal lobes of the brain. Patients with frontotemporal dementia typically have atrophy of the frontal and / or temporal lobes and can exhibit symptoms that include unusual behavior, emotional problems, difficulty communicating, and difficulty walking. Frontotemporal dementia is rare and tends to occur at a younger age than other types of dementia. Approximately 60% of people with this type of dementia are 45 to 64 years old.

[0062] Germ cell tumor: A type of tumor that begins in the cells that give rise to sperm or eggs (germ cells). Germ cell tumors can occur almost anywhere in the body and can be either benign or malignant.

[0063] Glioblastoma: A fast-growing type of central nervous system tumor that forms from glial tissue of the brain and spinal cord and has cells that look very different from normal cells. Glioblastoma usually occurs in adults and affects the brain more often than the spinal cord. Glioblastoma is also called GBM, glioblastoma multiforme, and grade IV astrocytoma.

[0064] Glioma: A cancer of the brain that begins in the glial cells, which are cells that surround and support nerve cells.

[0065] Hepatocellular carcinoma (HCC): A primary malignancy of the liver typically occurring in patients with inflammatory livers resulting from viral hepatitis, liver toxins or hepatic cirrhosis (often caused by alcoholism). HCC is also called malignant hepatoma.

[0066] Human endogenous retrovirus K (HERV-K): A group of human endogenous retroviruses (HERV) that are present in hundreds of copies in the human genome. HML-2 is a subtype of HERV-K.

[0067] Isolated: An “isolated” biological component, such as a nucleic acid (e.g., shRNA molecule), protein or organelle, has been substantially separated or purified away from other biological components in which the component occurs. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. 4239-1 10530-02

[0068] The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0069] Kennedy's disease: An inherited lower motor neuron disorder that affects men. The onset of symptoms varies, but usually begins between the ages of 20 and 40. Kennedy’s disease is also known as spinal and bulbar muscular atrophy (SBMA), bulbo-spinal muscular atrophy, or X-linked spinal and bulbar muscular atrophy. It is caused by mutations in the gene for the androgen (male sex hormone) receptor. Kennedy's disease is slowly progressive. Early symptoms include tremor of the hands when they are outstretched, muscle cramps with exertion, and fasciculations. Eventually, individuals develop weakness in their arms and legs. Weakness of the facial and tongue muscles may occur later in the disorder and often leads to difficulty swallowing, slurred speech, and repeated cases of pneumonia. Currently, there is no known cure for Kennedy's disease.

[0070] Medulloblastoma: A fast-growing type of cancer that forms in the cerebellum. Medulloblastomas tend to spread through the cerebrospinal fluid to the spinal cord or to other parts of the brain. They may also spread to other parts of the body, but this is rare. Medulloblastomas are most common in children and young adults. They are a type of central nervous system embryonal tumor.

[0071] Melanoma: A form of cancer that originates in melanocytes (cells that make the pigment melanin). Melanocytes are found primarily in the skin, but are also present in the bowel and eye. Melanoma in the skin includes superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna (melanoma). Any of the above types may produce melanin or can be amelanotic. Similarly, any subtype may show desmoplasia (dense fibrous reaction with neurotropism) which is a marker of aggressive behavior and a tendency to local recurrence. Other melanomas include clear cell sarcoma, mucosal melanoma and uveal melanoma.

[0072] Motor neuron disease: A group of progressive neurological disorders that destroy motor neurons, the cells that control skeletal muscle activity such as walking, breathing, speaking, and swallowing. This group includes diseases such as amyotrophic lateral sclerosis (ALS), progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease, and post-polio syndrome. Messages or signals from nerve cells in the brain (upper motor neurons) are typically transmitted to nerve cells in the brain stem and spinal cord (lower motor neurons) and then to muscles throughout the body. When signals from the lower motor neurons to the muscles are disrupted, the muscles begin to weaken and shrink in size (muscle atrophy or wasting). They may also start 4239-1 10530-02 to spontaneously twitch. These twitches, called fasciculations, can sometimes be seen or felt below the surface of the skin. When lower motor neurons cannot receive signals from upper motor neurons, it can cause muscle stiffness (spasticity) and overactive reflexes. This can make voluntary movements slow and difficult. Over time, individuals with a motor neuron disease may lose the ability to walk or control other movements. Motor neuron diseases are classified according to whether the loss of function is due to a genetic mutation (inherited) or sporadic (no family history); and whether they affect the upper motor neurons, lower motor neurons, or both. Inherited motor neuron diseases are usually caused by changes in a single gene. Though there are several types of motor neuron diseases, they all cause muscle weakness that gradually worsens over time and can lead to physical disability. In some people, these diseases are fatal. Weakness in muscles that control breathing can lead to respiratory insufficiency, a condition in which the lungs cannot properly take in oxygen or expel carbon dioxide. Symptoms may include breathlessness, shortness of breath that occurs while lying down, recurrent chest infections, disturbed sleep, poor concentration and / or memory, confusion, morning headaches, and fatigue. See, e.g., ninds.nih.gov / health- information / disorders / motor-neuron-diseases#.

[0073] Multifocal motor neuropathy (MMN): A rare, acquired motor neuropathy characterized by progressive asymmetric weakness without sensory loss. The arms are typically more affected than the leg. The majority of patients with MMN have serum IgM anti-ganglioside antibodies.

[0074] Neurological disorder: Any disorder that affects the central nervous system or the peripheral nervous system, such as the brain, spinal cord and nerves. Neurological disorders include, for example, motor neuron diseases (such as ALS), epilepsy, stroke, Alzheimer's disease, Parkinson’s disease, schizophrenia, muscular dystrophy, multiple sclerosis, and spinal cord injury.

[0075] Ovarian cancer: Cancer that forms in tissues of the ovary (one of a pair of female reproductive glands in which the ova, or eggs, are formed). Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells).

[0076] Pancreatic cancer: A disease in which malignant cells are found in the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer. The vast majority (-94%) of pancreatic cancers are exocrine tumors. Exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (IPMN), 4239-1 10530-02 and mucinous cystadenocarcinoma. In some examples, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also referred to as islet cell tumors, are classified by the type of hormones they produce. Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide) and nonfunctional islet cell tumor.

[0077] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature of the carrier 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. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, 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.

[0078] Post-polio syndrome (PPS): A motor neuron disease that usually occurs 15-40 years after a person has polio, an infectious viral disease. PPS is believed to be the result of deterioration of the motor neurons over many years that leads to loss of muscle strength and dysfunction. It is not contagious and only someone who has had polio can develop PPS. Not everyone who has had polio will develop PPS. The polio vaccine has essentially eradicated polio from the U.S. However, polio still exists in some countries, where cases of PPS still occur. PPS is rarely life-threatening, but the symptoms, which include muscle weakness, fatigue, atrophy, and scoliosis, can significantly interfere with a person’s ability to function independently.

[0079] Preventing, treating or ameliorating a disease: “Preventing” a disease (such as a motor neuron disease) refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease. 4239-1 10530-02

[0080] Primary lateral sclerosis (PLS): A motor neuron disease that affects only the upper motor neurons, causing difficulty and slowness in the movements of the arms, legs, and face. Symptoms include weakness, muscle stiffness and spasticity, clumsiness, slowing of movement, and problems with balance and speech. The disorder often affects the legs first, followed by the torso, arms and hands, and, finally, the muscles used for swallowing, speaking, and chewing. PLS is more common in men than in women, with an onset that generally occurs between age 40 and 60. PLS progresses slowly over years or even decades. There is no cure for PLS and the rate of symptom progression varies. Many people can walk without assistance early on, but most will need canes, walkers, wheelchairs, or other assistive devices to prevent falls and injuries as the disorder progresses.

[0081] Progressive bulbar palsy (PBP): A motor neuron disease, also known as progressive bulbar atrophy, that results from injury of the upper motor neurons in the brainstem or the lower motor neurons connected to the brainstem. The brainstem controls the muscles needed for swallowing, speaking, chewing, and other functions. PBP symptoms worsen over time and include trouble chewing, speaking, and swallowing. People with progressive bulbar palsy may also have weakness in the tongue and facial muscles, twitches, and a reduced gag reflex. They may also experience weakness in the arms or legs, but it is less noticeable than other symptoms. Because they have difficulty swallowing, people with PBP are at risk of choking and inhaling food and fluids, including saliva, into the lungs. They may also laugh or cry at inappropriate times, called pseudobulbar affect or pathological laughing and crying. Some symptoms of stroke and myasthenia gravis are similar to those of progressive bulbar palsy (e.g., slurring of the speech and choking) and must be ruled out prior to diagnosis. Many ALS experts consider PBP to be a form of ALS because the majority of individuals who begin with this form of the disease eventually develop more widespread motor neuron disease symptoms.

[0082] Progressive muscular atrophy (PMA): An uncommon subtype of ALS marked by slow but progressive damage to the lower motor neurons. It affects men more often than women, and usually symptoms begin later in life than typical ALS. People with PMA usually notice weakness in their hands or feet followed by other body regions. They may have weakness in the torso muscles and may have trouble breathing. Exposure to cold can worsen the person’s symptoms. Other symptoms may include muscle wasting or shrinking, clumsy hand movements, twitches, and muscle cramps.

[0083] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g. a gene). A promoter includes necessary nucleic acid sequences near the start site of 4239-1 10530-02 transcription. Typically, promoters are located near the genes they transcribe. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.

[0084] Prostate cancer: A malignant tumor, generally of glandular origin, of the prostate. Prostate cancers include adenocarcinomas and small cell carcinomas. Many prostate cancers express prostate specific antigen (PSA).

[0085] Recombinant: A recombinant nucleic acid, protein, or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In some aspects, a recombinant viral vector (such as recombinant AAV vector) includes a heterologous sequence (such as a shRNA coding sequence).

[0086] Schizophrenia: A chronic brain disorder that can include delusions, hallucinations, disorganized speech, trouble with cognition and lack of motivation.

[0087] Short hairpin RNA (shRNA): A sequence of RNA that makes a tight hairpin turn and can be used to silence gene expression via the RNAi pathway. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA.

[0088] Spinal muscular atrophy (SMA): A group of hereditary diseases that affect lower motor neurons. The most common form is caused by a mutated or missing gene known as the survival motor neuron gene 1 (SMN1), which causes the neurons to deteriorate, producing muscle weakness and wasting.

[0089] Spinal muscular atrophy with respiratory distress type 1 (SMARD1): A very rare form of SMA caused by mutations in the IGHMBP2 (immunoglobulin helicase p-binding protein 2) gene. Symptoms appear during infancy, between ages 6 weeks and 6 months. Children with SMARD1 suddenly may be unable to breathe due to diaphragm paralysis and may develop weakness in the muscles of their hands and feet.

[0090] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In some aspects, the subject has a disease or disorder associated with expression of HERV-K genes, such as a subject with a neurological disorder or cancer.

[0091] Therapeutically effective amount: The amount of agent, such as one or more shRNA molecules or vectors disclosed herein, alone or in combination with additional agents, sufficient to prevent, treat, reduce and / or ameliorate one or more symptoms and / or underlying causes of a disorder or disease, such as a motor neuron disease or cancer. The one or more 4239-1 10530-02 symptoms of the disease or disorder do not have to be completely eliminated for the composition to be effective.

[0092] Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more shRNA coding sequences (such as one or more of SEQ ID NOs: 1-24) and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like. Exemplary vectors include plasmids and viral vectors (such as lentiviral vectors and adeno- associated vectors).

[0093] III. Short Hairpin RNA Molecules and Vectors Targeting HERV-K

[0094] Disclosed herein are short hairpin RNA (shRNA) molecules targeting the human endogenous retrovirus K (HERV-K) Env gene. Although the shRNA molecules were designed to target the Env gene, the disclosed shRNA molecules are capable of blocking / inhibiting expression of the HERV-K Env, Gag and Pol genes. Further disclosed herein are recombinant viral vectors, such as AAV or lentiviral vectors, encoding the disclosed HERV-K-targeted shRNA molecules. The shRNA molecules and vectors can be used to inhibit expression of HERV-K genes in vivo, and thereby treat neurological disorders and tumors that are associated with HERV-K gene expression.

[0095] Provided herein are isolated shRNA molecules targeting a HERV-K nucleic acid molecule. In some aspects, the shRNA molecules target a region of the HERV-K Env gene (e.g., the HERV-K subtype HML-2 Env gene), such as a region of the Env gene set forth as any one of SEQ ID NOs: 25-48. In some aspects, the shRNA molecule is encoded by a nucleic acid sequence that includes any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, or includes any one of SEQ ID NOs: 1-24 with no more than one, no more than two, no more than three, no more than four, no more than five, no more than six, no more than seven, no more than eight, no more than nine, or no more than 10 nucleotide substitutions or deletions 4239-1 10530-02

[0096] (such as deletions at the 5' and / or 3' end of the sequence). In some examples, the shRNA molecule is encoded by a nucleic acid sequence consisting of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. In particular examples, the shRNA is encoded by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.

[0097] Also provided herein are vectors that include a coding sequence for a shRNA molecule targeting a HERV-K nucleic acid molecule. In some aspects, the shRNA molecules target a region of the HERV-K Env gene (<?.g., the HERV-K subtype HML-2 Env gene), such as a region of the Env gene set forth as any one of SEQ ID NOs: 25-48. In some aspects, the coding sequence for the shRNA molecule includes any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, or includes any one of SEQ ID NOs: 1-24 with no more than one, no more than two, no more than three, no more than four, no more than five, no more than six, no more than seven, no more than eight, no more than nine, or no more than 10 nucleotide substitutions or deletions (such as deletions at the 5' and / or 3' end of the sequence). In some examples, the coding sequence of the shRNA molecule consists of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. In particular examples, the shRNA is encoded by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.

[0098] In some aspects, the vector includes coding sequences for at least two different shRNA molecules, at least three different shRNA molecules, at least four different shRNA molecules, or at least five different shRNA molecules. In some examples, the vector includes coding sequences for two different shRNA molecules. In specific examples, the coding sequences of the two different shRNA molecules include or consist of SEQ ID NO: 2 and SEQ ID NO: 4. In other examples, the vector includes coding sequences for three different 4239-1 10530-02 shRNA molecules. In specific examples, the three different shRNA molecules include or consist of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

[0099] In some aspects, the vector is a viral vector. In some examples, the viral vector is an adeno-associated virus (AAV) vector. In some instances, the AAV vector is a self- complementary AAV vector. In particular examples, the AAV vector is an AAV9 vector, or another AAV serotype with tropism for the central nervous system (e.g., AAV1, AAV2, AAV4, AAV5, AAV7, AAV 10, or AAV11). In some examples, the AAV vector is an AAV9-pseudotyped AAV (e.g., an AAV2 vector pseudotyped with AAV9). Alternative AAV serotypes can also be selected based on the desired target tissue (see, e.g., Issa et al., Cells 12(5):785, 2023). For example, other AAV serotypes include AAV3, AAV6, AAV8, and AAV 12. In some examples, the AAV vector includes a promoter, such as but not limited to, a U6 promoter, a cytomegalovirus (CMV) promoter, or any other suitable promoter known to a skilled person. In specific examples, when the AAV vector includes coding sequences for multiple shRNA molecules, the vector contains a promoter (such as a U6 promoter) for each inserted shRNA coding sequence (see FIG. 6 and FIG. 7). In other examples, the viral vector is a lentiviral vector.

[0100] In alternative aspects, the shRNA(s) or the shRNA coding sequence(s) (or a plasmid vector containing the shRNA coding sequence(s)) is administered as part of a nanoparticle. In some aspects, the nanoparticles are lipid-based nanoparticles, such as but not limited to micelles, liposomes, solid lipid nanoparticles, polymeric nanoparticles, polymeric micelles, polymersomes, dendrimers, cyclodextrin polymers, and nanocapsules (see, e.g., Morales- Becerril et al., EXCLI J 21:1028-1052, 2022). In other aspects, the nanoparticles are inorganic nanoparticles, such as but not limited to, gold nanoparticles, magnetic nanoparticles, silica nanoparticles, carbon nanotubes, quantum dots, and ceramic nanoparticles (see, e.g., Morales-Becerril et al. , EXCLI 721 :1028-1052, 2022; and Moazzam et al., Mol Ther 32(2):284-312, 2024). In other aspects, the nanoparticles are protein-based nanoparticles, such as but not limited to, fibroin nanoparticles, human serum albumin nanoparticles, gliadin nanoparticles, gelatin nanoparticles, legumin nanoparticles, 30Kcl9 (a protein from silkworms) nanoparticles, lipoprotein nanoparticles, casein nanoparticles, encapsuling nanoparticles, and ferritin nanoparticles (see, e.g., Hong et al., Pharmaceutics 12(7):604, 2020; Kianfar, J Nanobiotechnology 19(1):159, 2021; Olshefsky et al., Bioconjugate Che i 33(11) :2018-2334, 2022). 4239-1 10530-02

[0101] Further provided are isolated host cells that include an shRNA (or its coding sequence) or a vector disclosed herein. In some aspects, the cell is a mammalian cell. In some examples, the mammalian cell is an HEK293 cell.

[0102] Also provided herein are compositions that include a pharmaceutically acceptable carrier and at least one isolated shRNA molecule, a vector, or a nanoparticle disclosed herein. In some aspects, the composition includes at least two isolated shRNA molecules, at least three isolated shRNA molecules, at least four isolated shRNA molecules, or at least five isolated shRNA molecules (or a vector or nanoparticle encoding or containing at least one, at least two, at least three, at least four, or at least five shRNA molecules). In some examples, the composition includes two different shRNA molecules, wherein the shRNA molecules have coding sequences that include or consist of SEQ ID NO: 2 and SEQ ID NO: 4. In other examples, the composition includes three different shRNA molecules, wherein the shRNA molecules have coding sequences that include or consist of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5. In other aspects, the composition includes a pharmaceutically acceptable carrier and a nucleic acid coding for at least one shRNA molecule disclosed herein. In some examples, the composition includes nucleic acids coding for two different shRNA molecules, wherein the nucleic acids include or consist of SEQ ID NO: 2 and SEQ ID NO: 4. In other examples, the composition includes nucleic acids coding for three different shRNA molecules, wherein the nucleic acid sequences include or consist of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

[0103] In some aspects, the composition is formulated for intravenous, oral, intranasal, subcutaneous, inhalation, or parenteral administration.

[0104] IV. Methods of Treatment

[0105] Expression of HERV-K genes is associated with several pathological conditions, including neurological disorders (e.g., ALS) and some types of cancer. Thus, the shRNA molecules and recombinant viral vectors having coding sequence(s) for one or more shRNA molecules, can be used in vivo to inhibit HERV-K gene expression and thereby treat diseases associated with HERV-K gene expression. In some aspects of the methods disclosed herein, the subject has a disease or disorder associated with expression of HERV-K genes (such as a neurological disorder or cancer). In some aspects, the method includes selecting a subject having a disease or disorder associated with expression of HERV-K genes (such as a neurological disorder or cancer). 4239-1 10530-02

[0106] Provided is a method of inhibiting expression of HERV-K gene expression in a subject by administering to the subject an effective amount of an shRNA molecule, vector, or composition disclosed herein, thereby inhibiting expression of HERV-K in the subject. In some aspects, the shRNA molecule, vector, or composition inhibits expression of the HERV- K Env, Pol and / or Gag gene.

[0107] Also provided is a method of treating a neurological disorder in a subject by administering to the subject a therapeutically effective amount of an shRNA molecule, vector, or composition disclosed herein, thereby treating the neurological disorder in the subject. In some aspects, the neurological disorder is a motor neuron disease, Alzheimer's disease, schizophrenia, or frontotemporal dementia. In some examples, the motor neuron disease is amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), spinal muscular atrophy with respiratory distress type 1 (SMARD1), congenital SMA with arthrogryposis, progressive bulbar palsy (PBP), primary lateral sclerosis, progressive muscular atrophy (PMA), Kennedy’s disease, post-polio syndrome (PPS), or multifocal motor neuropathy (MMN). In some aspects, the subject is administered one or more additional therapies suitable for treating the neurological disorder and / or alleviating one or more symptoms of the disorder. In some aspects, the subject with a neurological disorder is administered one or more additional therapies to treat the disease and / or reduce the symptoms of the disease. For example, a subject with ALS can be further treated with riluzole (also sold as RILUTEK, EXSERVAN or TIGLUTIK) or edaravone (also sold as RADICAVA).

[0108] Further provided is a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of an shRNA molecule, vector, or composition disclosed herein, thereby treating the cancer in the subject. In some aspects, the cancer expresses a HERV-K Env, Gag, or Pol transcript or protein. In some aspects, the cancer is a brain tumor. In some examples, the brain tumor is medulloblastoma, atypical teratoid rhabdoid tumor, glioblastoma, glioma, or another developmental tumor of the brain. In other aspects, the cancer is breast cancer, prostate cancer, melanoma, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, or a germ cell tumor. In some aspects, the subject is administered one or more additional anti-cancer agents or therapies (such as surgical resection of a tumor or radiation therapy). Any suitable anti-cancer agent can be administered in combination with the compositions disclosed herein, such as administered prior to, concurrently with, or following administration of the disclosed compositions. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating 4239-1 10530-02 antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

[0109] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0110] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0111] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0112] Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

[0113] Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571 , Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

[0114] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or 4239-1 10530-02

[0115] Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0116] In some aspects of the disclosed methods, the shRNA, vector or composition is formulated for intravenous administration. In other aspects, the shRNA, vector or composition is formulated for oral, inhalation, or parenteral administration.

[0117] In some aspects of the disclosed methods, the subject is administered an AAV vector (such as a self-complementary AAV vector) encoding one more HERV-K-targeted shRNA molecules. A suitable dose of AAV vector can be determined by a skilled person based on a variety of factors, including the age, weight, gender, and general health of the subject, as well as the disease or disorder to be treated. In some aspects, the AAV is administered at a dose of about 1 x 1010to about 1 x 1014viral particles (vp) / kg. In some examples, the AAV is administered at a dose of about 1 x 1011to about 8 x 1013vp / kg or about 1 x 1012to about 8 x 1013vp / kg. In other examples, the AAV is administered at a dose of about 1 x 1013to about 6 x 1013vp / kg. In specific non-limiting examples, the AAV is administered at a dose of at least about 1 x 1010, at least about 5 x 1010, at least about 1 x 1011, at least about 5 x 1011, at least about 1 x 1012, at least about 5 x IO12, at least about 1 x 1013, at least about 5 x 1013, or at least about 1 x 1014vp / kg. In other non-limiting examples, the AAV is administered at a dose of no more than about 1 x 1010, no more than about 5 x 1010, no more than about 1 x 1011, no more than about 5 x 1011, no more than about 1 x 1012, no more than about 5 x 1012, no more than about 1 x 1013, no more than about 5 x 1013, or no more than about 1 x 1014vp / kg. In one non-limiting example, the AAV is administered at a dose of about 1 x 1012vp / kg. In another non-limiting example, the AAV is administered at a dose of about 1 x 1011vp / kg. The AAV can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0118] EXAMPLES

[0119] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0120] Example 1: Construction and testing of AAV vectors for shRNA against HERV-K Env

[0121] Using Invitrogen BLOCK-iT™ RNAi Designer, shRNAs were designed to target a consensus HERV-K Env sequence. Table 1 provides the coding sequences for each shRNA. Table 2 shows the HERV-K gene sequence targeted by the designed shRNA molecules. 4239-1 10530-02

[0122] Table 1: shRNA coding sequences

[0123] Table 2. HERV-K Env sequences targeted by shRNA 4239-1 10530-02

[0124] The shRNA coding sequences (Table 1) were cloned into an AAV vector with RNA polymerase 111 promoter for human U6 snRNA (U6 promoter). To test the effectiveness of the encoded shRNA molecules for blocking HERV-K expression, HEK293 cells were co- transfected with a HERV-K plasmid and an individual shRNA vector. The shRNA molecules having the coding sequences of SEQ ID NOs: 1-14 were tested in this study and compared to a previously developed HERV-K-targeted shRNA (shRNA 1000). Env mRNA was measured by PCR at 24, 48, and 72 hours post-transfection. All of the shRNA molecules showed at least some blocking effect, with shRNAs targeting the 5 ’ end of the Env sequence being the most effective (FIGS. 1A-1C). A subset of the shRNAs (231, 235, 238, 286, 343, 470, 878, 1000 and 1033) were further tested for blocking other HERV-K genes. As shown 4239-1 10530-02 in FIG. 2, although the shRNAs target the Env sequence, they blocked all HERV-K genes (Gag, Pol, and Env).

[0125] Example 2: Tandem shRNA vector construction and testing

[0126] To further increase the efficiency of blocking HERV-K expression, three shRNAs (shRNA235, shRNA286, and shRNA343; SEQ ID NOs: 2, 4 and 5, respectively) were selected for generating a tandem shRNA vector (FIG. 6). Two or three independent shRNA expression cassettes were cloned in tandem in a single AAV vector. The tandem vectors were compared to individual shRNA for blocking HERV-K genes. As shown in FIG. 3, three tandem shRNAs (shRNA 235-286-343) were the most effective at blocking expression of all HERV-K genes. Western blot analysis further confirmed that the three tandem shRNA vector significantly blocked Env protein expression (FIGS. 4A-4C).

[0127] Example 3: Tandem shRNA against HERV-K from different chromosomal loci

[0128] Since there are many copies of HERV-K in the human genome, it is important to target as many HERV-K loci as possible, especially those with Env expression. It is believed that Env expression is responsible for some motor neuron diseases and for some types of tumorigeneses. HERV-K sequences with an intact Env open reading frame from selected loci (5q33.3, 6ql4. 1 , 7p22.1, 12ql4. 1 , and 19pl2b) were synthesized and cloned into a pcDNA3. 1 plasmid. The plasmid was co-transfected with the tandem shRNA vector and tested for viral gene expression. As shown in FIG. 5, the tandem shRNA vector blocked gene expression from all the tested HERV-K loci.

[0129] Example 4: Self-complementary AAV vector

[0130] A self-complementary AAV vector (AAV-2 pseudotyped with AAV-9) with three shRNAs (shRNA235, shRNA286, and shRNA343) was generated, which is referred to herein as sHscA-9 (FIG. 7). The plasmid was modified to introduce a complementary strand of each of the shRNAs in the plasmid. The ability of the DNA to fold upon itself leads to a double stranded DNA without the need for the cell to express these genes, leading to better efficacy (see FIG. 8). Each of the shRNAs with its complementary strand is regulated by a U6 promoter which ensures equal production of each of the shRNAs. To increase the yield of the double stranded DNA, this strategy also requires deleting the terminal resolution site sequence from one ITR, such that the Rep protein cannot generate the essential ssDNA nick (McCarty etal., Gene Ther 10(26):2112-2118, 2003; Wang et al., Gene Ther 10(26):2105- 4239-1 10530-02

[0131] 2111, 2003). The replication complex initiated at the other ITR then copies through the hairpin and back toward the initiating end. Replication proceeds to the end of the template molecule, leaving a dsDNA inverted repeat with a wild-type ITR at each end and the mutated ITR in the middle. This dimeric inverted repeat can then undergo normal rounds of replication from the two wildtype ITR ends. Each displaced daughter strand includes a ssDNA inverted repeat with a complete ITR at each end and a mutated ITR in the middle. Packaging into the AAV capsid ensues from the 3' end of the displaced strand. Previous studies showed that production of self-complementary AAV (scAAV) from constructs with one mutated ITR typically yields >90% dimeric genomes (McCarty, Mol Ther 16(10): 1648- 1656, 2008).

[0132] To test the effect of sHscA-9 on HML-2 transcripts, Tera-1 cells were seeded in 12- well plates at a density of IxlO5cells per well and infected with sHscA-9 (scAAV) or sHA-9 (ssAAV) in serum free media. An AAV-9 plasmid with the mutated ITR similar to the scAAV but without the shRNA was used as a control (control AAV). RNA was extracted from the cells at 72 hours and analyzed by RT-PCR for each of the HML-2 transcripts. The results are shown in FIG. 8. The decrease in HML-2 transcripts was more robust using scAAV relative to AAV.

[0133] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239-1 10530-02CLAIMS1. An isolated short hairpin RNA (shRNA) molecule targeting a human endogenous retrovirus K (HERV-K) nucleic acid molecule, wherein the shRNA molecule is encoded by a nucleic acid sequence comprising any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

2. The isolated shRNA molecule of claim 1, wherein the shRNA molecule is encoded by a nucleic acid sequence consisting of any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

3. A vector comprising a coding sequence for a short hairpin RNA (shRNA) molecule targeting a human endogenous retrovirus K (HERV-K), wherein the coding sequence for the shRNA molecule comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

4. The vector of claim 3, wherein the coding sequence of the shRNA molecule consists of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.4239-1 10530-025. The vector of claim 3 or claim 4, comprising coding sequences for at least two different shRNA molecules.

6. The vector of claim 5, wherein the coding sequences of the at least two different shRNA molecules comprise or consist of SEQ ID NO: 2 and SEQ ID NO: 4.

7. The vector of claim 3 or claim 4, comprising coding sequences for at least three different shRNA molecules.

8. The vector of claim 7, wherein coding sequences of the at least three different shRNA molecules comprise or consist of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

9. The vector of any one of claims 3-8, wherein the vector is an adeno- associated virus (AAV) vector or a lentiviral vector.

10. The vector of claim 9, wherein the AAV vector is an AAV9 vector or an AAV9-pseudotyped vector.

11. The vector of claim 9 or claim 10, wherein the AAV vector is a self- complementary AAV vector.

12. An isolated host cell comprising the shRNA of claim 1 or claim 2, or the vector of any one of claims 3-11.

13. The isolated cell of claim 12, wherein the cell is a mammalian cell.

14. A composition comprising a pharmaceutically acceptable carrier and at least one isolated shRNA molecule of claim 1 or claim 2, or the vector of any one of claims 3-11.

15. The composition of claim 14, comprising at least two isolated shRNA molecules, wherein the coding sequences of the at least two shRNA molecules comprise or consist of SEQ ID NO: 2 and SEQ ID NO: 4.4239-1 10530-0216. The composition of claim 14, comprising at least three isolated shRNA molecules, wherein the coding sequences of the at least three shRNA molecules comprise or consist of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

17. A method of inhibiting expression of human endogenous retrovirus K (HERV- K) gene expression in a subject, comprising administering to the subject an effective amount of the shRNA molecule of claim 1 or claim 2, the vector of any one of claims 3-11, or the composition of any one of claims 14-16, thereby inhibiting expression of HERV-K in the subject.

18. The method of claim 17, wherein the shRNA molecule, vector or composition inhibits expression of the HERV-K Env, Pol and / or Gag gene,19. A method of treating a neurological disorder in a subject, comprising administering to the subject a therapeutically effective amount of the shRNA molecule of claim 1 or claim 2, the vector of any one of claims 3-11, or the composition of any one of claims 14-16, thereby treating the neurological disorder in the subject.

20. The method of claim 19, wherein the neurological disorder is a motor neuron disease, Alzheimer's disease, schizophrenia, or frontotemporal dementia.

21. The method of claim 20, wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), spinal muscular atrophy with respiratory distress type 1 (SMARD1), congenital SMA with arthrogryposis, progressive bulbar palsy (PBP), primary lateral sclerosis, progressive muscular atrophy (PMA), Kennedy’s disease, post-polio syndrome (PPS), or multifocal motor neuropathy (MMN).

22. A method of treating a cancer in a subject, wherein the cancer expresses a HERV-K Env, Gag, or Pol transcript or protein, comprising administering to the subject a therapeutically effective amount of the shRNA molecule of claim 1 or claim 2, the vector of any one of claims 3-11, or the composition of any one of claims 14-16, thereby treating the cancer in the subject.

23. The method of claim 22, wherein the cancer is a brain tumor.4239-1 10530-0224. The method of claim 23, wherein the brain tumor is medulloblastoma, atypical teratoid rhabdoid tumor, glioblastoma, or glioma.

25. The method of claim 22, wherein the cancer is breast cancer, prostate cancer, melanoma, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, or a germ cell tumor.

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