WO2025212926A1

WO2025212926A1 - Aav-mediated rhoa knockdown

Info

Publication number: WO2025212926A1
Application number: PCT/US2025/023010
Authority: WO
Inventors: Meng GAO; Tapan Sharma; Bo Tian; Haijiang Lin; Jun Xie; Guangping Gao; Phillip TAI
Original assignee: University of Massachusetts Amherst
Current assignee: University of Massachusetts Amherst
Priority date: 2024-04-05
Filing date: 2025-04-03
Publication date: 2025-10-09
Anticipated expiration: 2026-10-05

Abstract

Provided herein are inhibitory nucleic acids (e.g., artificial microRNAs) and rAAV vectors targeting Ras homolog family member A (RhoA) and uses of the same for treating conditions related to increased intraocular pressure (IOP) (e.g., glaucoma).

Description

AAV-MEDIATED RHOA KNOCKDOWN

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of United States Provisional Application Serial Number 63/575,157, filed April 5, 2024, and entitled “AAV-MEDIATED RHOA KNOCKDOWN,” the entire contents of which are herein incorporated by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (U012070200WO00-SEQ-KZM.xml; Size: 110,608 bytes; and Date of Creation: April 1, 2025) is herein incorporated by reference in its entirety.

BACKGROUND

Glaucoma is one of the leading causes of adult blindness and affects about 80 million individuals globally. In primary open angle glaucoma (POAG), a key high-risk factor that aggravates blindness in these subjects is the accumulation of excess aqueous humor. Aqueous humor results in increased ocular pressure and causes damage to the optic nerve, leading to loss of sight. RhoA belongs to the ubiquitously expressed family of small GTPases. Activated GTP-bound RhoA can associate with and activate Rho-associated serine-threonine kinases (ROCK1/2), which are capable of phosphorylating a number of substrates, including myosin light chain (MLC), myosin phosphatase target subunit (MYPT-1), and LIM kinases 1/2. Phosphorylation of these substrates results in increased actin polymerization, which in turn increases cellular contraction. One major hindrance to the outflow of aqueous humor is increased contraction of the trabecular meshwork cell layer of the eye.

SUMMARY

Aspects of the disclosure relate to compositions and methods useful for treating diseases and disorders associated with increased intraocular pressure (e.g., glaucoma). In some aspects, the disclosure provides nucleic acids (e.g., rAAV vectors) configured to express transgenes that inhibit (e.g., decrease) expression of RhoA. In some aspects, the transgenes encode artificial microRNAs (amiRNAs) targeting RhoA. A transgene generally refers to a nucleic acid encoding one or more gene products (e.g., one or more functional RNAs, such as amiRNAs). Accordingly, in some aspects, the disclosure provides isolated nucleic acids comprising a transgene encoding an inhibitory nucleic acid targeting a RhoA messenger RNA (mRNA), wherein the transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some aspects, the disclosure provides isolated nucleic acids comprising an inhibitory nucleic acid having a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some embodiments, the inhibitory nucleic acid is an artificial microRNA (amiRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the inhibitory nucleic acid is an artificial microRNA (amiRNA). In some embodiments, the amiRNA comprises a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-11. In some embodiments, the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, or 99% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 19, 24, or 25. In some embodiments, the inhibitory nucleic acid is an amiRNA comprising a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 9, or 10.

In some embodiments, the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50. In some embodiments, the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

In some embodiments, the inhibitory nucleic acid is complementary to a segment of a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50. In some embodiments, a segment of nucleic acid comprises 2-24 contiguous nucleotides. In some embodiments, the inhibitory nucleic acid is complementary to a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

In some embodiments, the nucleic acid encoding the inhibitory nucleic acid is operably linked to a promoter. In some embodiments, the promoter is a trabecular mesh work- specific promoter. In some embodiments, the trabecular meshwork- specific promoter is a Chitinase-3- like 1 (Ch3Ll) promoter. In some embodiments, the Ch3Ll promoter comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the Ch3Ll promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12.

In some embodiments, the ITRs are AAV2 ITRs.

In some aspects, the disclosure provides vectors comprising an isolated nucleic acid as described herein. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated virus vector, a lentiviral vector, a retroviral vector, or a Baculovirus vector.

In some aspects, the disclosure provides compositions comprising an isolated nucleic acids or vector as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In some aspects, the disclosure provides host cells comprising isolated nucleic acids or vectors as described herein. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a trabecular meshwork cell.

In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising (i) an isolated nucleic acid as described herein; and (ii) an adeno-associated virus (AAV) capsid protein. In some embodiments, the capsid protein has a tropism for ocular tissue. In some embodiments, the capsid protein is of a serotype selected from AAV2, AAV5, AAV8, AAV9, AAVrhlO, and a variant of any of the foregoing. In some embodiments, the capsid protein is an AAV2 capsid protein.

In some aspects, the disclosure provides methods for preventing or treating glaucoma in a subject, the methods comprising administering to the subject an isolated nucleic acid, a vector, a composition, or an rAAV as described herein. In some embodiments, the glaucoma is primary open angle glaucoma (POAG). In some embodiments, the administering reduces intraocular pressure (IOP) in the subject. In some aspects, the disclosure provides methods of decreasing intraocular pressure (IOP) in a subject, the methods comprising administering to the subject an isolated nucleic acid, a vector, a composition, or an rAAV as described herein. In some embodiments, the administering is done via ocular injection. In some embodiments, the ocular injection is an intracameral injection.

In some embodiments, the administering decreases RhoA expression in ocular tissue of the subject. In some embodiments, the administering decreases Rho-associated serine/threonine kinase (ROCK) signaling in ocular tissue of the subject. In some embodiments, the administering decreases accumulation of aqueous humor (AH) in an eye of the subject. In some embodiments, the administering decreases contractility of a trabecular meshwork cell in an eye of the subject.

In some aspects, the disclosure provides methods of treating glaucoma in a subject, the methods comprising administering to the subject an isolated nucleic acid comprising a transgene encoding an inhibitory nucleic acid targeting a RhoA messenger RNA (mRNA), wherein the transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some aspects, the disclosure provides methods of treating glaucoma in a subject, the methods comprising administering to the subject an isolated nucleic acid comprising an inhibitory nucleic acid having a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some embodiments, the inhibitory nucleic acid is an artificial microRNA (amiRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the inhibitory nucleic acid is an artificial microRNA (amiRNA). In some embodiments, the amiRNA comprises a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-11. In some embodiments, the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 19, 24, or 25. In some embodiments, the inhibitory nucleic acid is an amiRNA comprising a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 9, or 10.

In some embodiments, the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50. In some embodiments, the RhoA mRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

In some embodiments, the inhibitory nucleic acid is complementary to a segment of a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50. In some embodiments, the segment of nucleic acid comprises 2-24 continuous nucleotides. In some embodiments, the inhibitory nucleic acid is complementary to a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

In some embodiments, the ITRs are AAV2 ITRs.

In some embodiments, the glaucoma is primary open angle glaucoma (POAG).

In some embodiments, the administering is done via ocular injection. In some embodiments, the ocular injection is an intracameral injection.

In some embodiments, the administering reduces intraocular pressure (IOP) in the subject. In some embodiments, the administering decreases RhoA expression in ocular tissue of the subject. In some embodiments, the administering decreases Rho-associated serine/threonine kinase (ROCK) signaling in ocular tissue of the subject. In some embodiments, the administering decreases accumulation of aqueous humor (AH) in an eye of the subject. In some embodiments, the administering decreases contractility of a trabecular meshwork cell in an eye of the subject.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1D demonstrate in vitro screening of unique artificial microRNAs (amiRs) targeting RhoA. FIG. 1A shows a schematic representation of designed amiR seed sequences (amiR-l-amiR-8 and amiR-10-amiR12) within the coding sequence (CDS) and the 3’ untranslated region (3’ UTR) of mouse and human RhoA transcripts (msRhoA and hsRhoA, respectively). FIG. IB shows an illustration of the workflow of luciferase assays used to assess RhoA knockdown efficiency. psiCheck reporter plasmids were co-transfected with amiR-RhoA- EGFP expressing constructs in kidney (HEK293) and trabecular meshwork (TM1) cells. FIGs. 1C-1D show the relative knockdown of mouse and human rhoa using amiR-RhoA test sequences (amiR-l-amiR-8 and amiR-10-amiR12) in HEK293 (FIG. 1C) and TM1 (FIG. ID) cells.

FIGs. 2A-2B demonstrate in vitro screening of unique amiRs driven by a trabecular meshwork-specific promoter. FIG. 2A shows fluorescence microscopy of EGFP driven by a Ch3Ll or CB6 promoter in TM1 and Neuro-2A cells. The top row shows expression of EGFP. The bottom row shows brightfield images of the cells. FIG. 2B shows the results of a psiCheck luciferase assay evaluating the mouse and human rhoa knockdown efficiency in TM1 cells transfected with Ch3Ll promoter-driven amiR4, amiRlO, and amiRl l. Empty vector was used as control.

FIG. 3 demonstrates in vitro knockdown of RhoA protein using amiR-RhoA constructs. FIG. 3 top panel shows a Western Blot of lysates from TM1 cells transfected with Ch3Ll promoter-driven amiR4, amiRlO, and amiRl l. Untransfected cells (UTC) and cells transfected with empty vector (Emp V) were used as control. The top row was immunoblotted for RhoA. The bottom row was immunoblotted for GAPDH and used as loading control. FIG. 3 bottom panel shows the quantification of the Western Blot band intensities plotted as RhoA to GAPDH.

FIGs. 4A-4C show experimental results assessing the processing precision of the unique amiRs targeting RhoA. FIG. 4A shows the guide-to-passenger (G/P) ratios for the top three amiR designs (amiR4, amiRlO, and amiRl 1) as a measure of efficacy of RNAi design. FIG. 4B shows the end processing of mature amiRs at both the 5’ (top) and 3’ (bottom) ends, demonstrating over 90% accuracy of processing in all amiR designs. FIG. 4C demonstrates expression analysis of endogenous miRNAs in TM1 cells transduced with AAV2-packaged amiR4, amiRlO, or amiRl l.

FIGs. 5A-5B illustrate the mechanism for open-angle glaucoma. FIG. 5A illustrates the increased intraocular pressure in the eyes of patients with glaucoma. FIG. 5B illustrates the production of aqueous humor (AH) by the ciliary body of the eye and the outflow of AH through layers of the trabecular meshwork and Schlemm’s Canal.

FIG. 6 shows a schematic of how the activation of RhoA may result in increased intraocular pressure. Activated (GTP-bound) RhoA triggers the phosphorylation of Rho- associated kinases 1/2 (ROCK1/2), which acts on substrates such as myosin light chain (MLC) and myosin phosphatase target subunit (MYPT-1). These events result in increased actin polymerization, decreased aqueous humor outflow, and increased intraocular pressure.

FIG. 7 shows the intraocular pressure in the eyes of mice administered increasing doses of rAAV comprising a transgene encoding amiRlO under the control of the ubiquitous CB6 promoter (AAV2.CB6. amiRlO). Intracameral injection of AAV2.CB6. amiRlO increased aqueous humor outflow and decreased intraocular pressure in normal ocular tension mice. rAAV comprising a transgene encoding EGFP was used as control. Significance was defined by comparisons to baseline (i.e., pre-treatment values) among different groups. *, p<0.05; **, p<0.01; ***, p<0.001. DETAILED DESCRIPTION

Aspects of the disclosure relate to compositions and methods for treating conditions or diseases associated with increased intraocular pressure (e.g., glaucoma). In some embodiments, a disease associated with intraocular pressure is glaucoma (e.g., primary open angle glaucoma). The disclosure is based, in part, on compositions (e.g., compositions comprising one or more nucleic acid sequences, vectors, rAAVs, etc.) that reduce the expression of RhoA (e.g., via reduction of rhoa RNA transcripts). In some embodiments, the compositions disclosed herein reduce the expression of a human or a mouse RhoA. In some embodiments, the compositions disclosed herein decrease intraocular pressure, decrease contraction of trabecular meshwork cells, and/or decrease Rho-associated kinase 1/2 (R0CK1/2) activity.

Compositions and methods for delivering a nucleic acid (e.g., a nucleic acid encoding an inhibitory nucleic acid, such as an artificial microRNA (amiRNA), microRNA (miRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), etc.) to a subject are provided in the disclosure. The compositions typically comprise an isolated nucleic acid encoding one or more nucleic acid sequences (also referred to as one or more transgenes) (e.g., an inhibitory nucleic acid) capable of modulating a gene encoding RhoA (e.g., RHOA) and/or treating a disease associated with increased intraocular pressure (e.g., glaucoma). For example, in some embodiments, a nucleic acid of the disclosure reduces expression of a target protein (e.g., RhoA) in the eye of a subject with increased ocular pressure (e.g., a subject with glaucoma).

Glaucoma

Glaucoma is a chronic and progressive group of optic neuropathies affecting more than 80 million people globally. It is associated with death of retinal ganglion cells, resulting in characteristic cupping or degeneration of the optic nerve head and loss of peripheral vision (Kwon et al., N Engl J Med. 2009 Mar 12;360(l l): 1113-24). Primary open angle glaucoma (POAG) is one of the most common types of glaucoma; POAG is clinically characterized by an open and normal anterior iridocorneal chamber angle and often occurs with increased intraocular pressure (IOP). While there are many postulated mechanisms of retinal ganglion cell damage in glaucoma, the exact etiology of POAG remains obscure. Well-recognized risk factors associated with POAG include, but are not limited to, elevated IOP, age, family history, gender, ethnicity, central corneal thickness, and myopia.

Maintenance of IOP is a key factor for sustaining eye health. IOP is maintained primarily by changes in the aqueous humor outflow resistance, which is thought to reside predominantly within the cribriform or juxtacanalicular (JCT) region of the trabecular meshwork (TM) and the inner wall of Schlemm’s canal (see, e.g., FIGs. 5A-5B). In POAG, sustained increases in the trabecular outflow resistance commonly result in elevated IOP, which is thought to occur at least in part by increased trabecular meshwork contractility, changes in extracellular matrix composition, and decreased pore density in the inner wall of Schlemm’s canal. Studies have indicated that reduction of IOP in glaucoma can slow damage to the optic nerve and preserve vision.

Currently, there are multiple options a physician may use to treat glaucoma. Traditionally, management of glaucoma starts with medical treatment (e.g., use of prescription drugs such as prostaglandin analogs, beta-blockers, alpha agonists, carbonic anhydrase inhibitors, miotic agents, etc.), and if necessary, proceeds to laser or incisional surgery. Recently, a class of ocular hypotensive drugs, rho kinase (ROCK) inhibitors, have been applied to decrease IOP by inhibiting ROCK, a ubiquitous downstream effector protein that regulates the cell cytoskeleton. ROCK inhibitors, which work by targeting ROCK’s activator, RhoA, are thought to directly affect the resistance of aqueous humor outflow by regulating the contractility of the trabecular meshwork and Schlemm’s canal cells. As used herein, “contractility” of a cell refers to the ability of a cell to change its size or shape through contraction of its cytoskeleton (e.g., actin or myosin filaments) and associated motor proteins (e.g., myosin). Cell contractility is an essential process required for homeostasis of cell movement, division, and maintenance of cell structure. In some embodiments, cell contractility is regulated through RhoA signaling. In some embodiments, cell contractility is regulated through ROCK (e.g., R0CK1/2) signaling.

Thus, in some aspects, administration of the compositions, nucleic acid sequences, isolated nucleic acids, and/or rAAVs of the present disclosure are useful for treating a subject having or suspected of having glaucoma (e.g., POAG) and/or its associated symptoms. In some embodiments, administration of the compositions, nucleic acid sequences, isolated nucleic acids, and/or rAAVs of the present disclosure reduces IOP by reducing ROCK activity through decreasing the availability of its activator, RhoA. In some embodiments, administration of the compositions, nucleic acid sequences, isolated nucleic acids, and/or rAAVs of the present disclosure reduces cell contractility of the trabecular meshwork and/or Schlemm’s canal. In some embodiments, administration of the compositions, nucleic acid sequences, isolated nucleic acids, and/or rAAVs of the present disclosure increase aqueous humor outflow. In some embodiments, administration of the compositions, nucleic acid sequences, isolated nucleic acids, and/or rAAVs of the present disclosure prevents or slows blindness. In some embodiments, administration of the compositions, nucleic acid sequences, isolated nucleic acids, and/or rAAVs of the present disclosure prevents or slows ocular nerve damage.

As used herein, the term “treating” refers to the application or administration of a composition as described herein to a subject, who has a disease associated with IOP, such as glaucoma (e.g., POAG), or a predisposition toward a disease associated with IOP, such as glaucoma (e.g., POAG), with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease.

Alleviating a disease includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease (such as a disease associated with IOP) means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

"Development" or "progression" of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a disease associated with IOP, such as glaucoma (e.g., POAG), includes initial onset and/or recurrence.

In some embodiments, the therapeutic methods as disclosed in this section comprise administering to a subject an isolated nucleic acid, a recombinant AAV (rAAV), a recombinant gene editing complex, or a vector, comprising a transgene as disclosed herein. An rAAV may comprise a modification that promotes its targeting to ocular tissues. In some embodiments, the therapeutic methods as disclosed herein comprise administering to a subject a rAAV comprising a capsid protein and an isolated nucleic acid encoding an inhibitory nucleic acid. The rAAV may comprise an inhibitory nucleic acid (e.g., siRNA, shRNA, miRNA, or amiRNA). The inhibitory nucleic acid may modulate (e.g., decrease) expression of a target gene associated with IOP. In some embodiments, the rAAV or isolated nucleic acid comprises a transgene encoding an artificial microRNA that targets a gene associated with glaucoma or the development of glaucoma. In some embodiments, the target gene is RhoA (e.g., RHOA).

IOP may be inhibited by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive) using methods of the present disclosure. Symptoms in a subject having or suspected of having IOP may be inhibited by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive) using methods of the present disclosure.

Inhibitory Nucleic Acids

In some embodiments, the present disclosure provides a nucleic acid comprising an inhibitory nucleic acid sequence targeting a RHOA gene transcript (e.g., a gene transcript encoded by NCBI Gene ID: 387 or 11848), which encodes Ras homolog family member A (RhoA). In some embodiments, the present disclosure provides inhibitory nucleic acids (e.g., amiRNA, miRNA, dsRNA, siRNA, shRNA, etc.). In some embodiments, an inhibitory nucleic acid sequence comprises a region of complementarity with a RHOA mRNA transcript. In some embodiments, a RHOA mRNA transcript comprises or consists of the nucleic acid sequence set forth in NCBI Reference Sequence: NM_001664.4, NM_001313941.2, NM_001313943.2, NM_001313944.2, NM_001313945.2, NM_001313946.2, NM_001313947.2, NM_001313961.1, NM_001313962.1, or NM_016802.5. In some embodiments, a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in NCBI Reference Sequence: NM_001664.4, NM_001313941.2, NM_001313943.2, NM_001313944.2, NM_001313945.2, NM_001313946.2, NM_001313947.2, NM_001313961.1, NM_001313962.1, or NM_016802.5. In some embodiments, a RHOA mRNA transcript comprises or consists of the nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50. In some embodiments, a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50.

Generally, an inhibitory nucleic acid specifically binds to (e.g., hybridizes with) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous bases of a target gene (e.g., a RhoA gene) or a messenger RNA encoded by a target gene (e.g., a RhoA mRNA). As used herein “continuous bases” refers to two or more nucleotide bases that are covalently bound (e.g., by one or more phosphodiester bond, etc.) to each other (e.g., as part of a nucleic acid molecule). In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript specifically binds to (e.g., hybridizes with) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous bases of a RHOA transcript. In some embodiments, an inhibitory nucleic acid is complementary to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous bases of a target gene (e.g., a RhoA gene) or a messenger RNA encoded by a target gene (e.g., a RhoA mRNA).

As used herein, the term “complementary” refers to the ability of a pair of nucleotides or a pair of nucleic acid sequences (e.g., an inhibitory nucleic acid of the disclosure and a target mRNA sequence) to bind to one another (e.g., using hydrogen bond pairing between two nucleotides). Two nucleic acid sequences or nucleic acid strands are “complementary” to one another if they base-pair, or bind, to each other to form a double- stranded nucleic acid molecule via Watson-Crick interactions and non-Watson-Crick base pairing (also referred to as hybridization). Binding to form a double-stranded nucleic acid molecule can refer to an association between at least two strands due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions (e.g., under physiological conditions). Non- Watson-Crick base pairing includes wobble base pairing and Hoogsteen base pairing. In some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine- type bases (T) or uracil-type bases (U); cytosine-type bases (C) are complementary to guanosine-type bases (G); and universal bases such as 3-nitropyrrole, 5-nitroindole, or inosine (I) can hybridize to and are considered complementary to any A-type, C-type, U-type, G-type, or T-type bases. In some embodiments, two nucleic acid sequences are complementary to one another if at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nucleobases across the length of one of the sequences are base-paired to nucleobases of the other sequence. Thus, in some embodiments, a sequence within an inhibitory nucleic acid is complementary to a target sequence if at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the nucleobases across the length of said sequence are base-paired to nucleobases of the target sequence. In some embodiments, an inhibitory nucleic acid comprises a sequence that is sufficiently complementary to a cognate target nucleotide sequence such that the inhibitory nucleic acid is capable of hybridizing to the cognate target nucleotide sequence (e.g., under physiological conditions such as the conditions in a cell). In some embodiments, an inhibitory nucleic acid oligonucleotide comprises a sequence that contains 1, 2, 3, 4, or 5 nucleobase mismatches with its cognate target nucleotide sequence. For example, an inhibitory nucleic acid may comprise a sequence of 15-25 nucleotides that contains 1, 2, 3, 4, or 5 nucleobase mismatches with its cognate target nucleotide sequence.

In some embodiments, an inhibitory nucleic acid that targets and/or binds to (e.g., is complementary to) a RHOA transcript (e.g., RHOA mRNA) comprises one or more inhibitory nucleic acid sequences provided in Table 1. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises one or more inhibitory nucleic acid sequences set forth in any one of SEQ ID NOs: 16-26. In some embodiments, an inhibitory nucleic acid comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 19, 24, and/or 25. In some embodiments, an inhibitory nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, an inhibitory nucleic acid comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some embodiments, an inhibitory nucleic acid comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 19, 24, and/or 25. In some embodiments, an inhibitory nucleic acid comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, an inhibitory nucleic acid comprises at least 8, at least 10, at least 12, or at least 15 contiguous nucleotides of the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26.

An inhibitory nucleic acid that targets a RhoA transcript (e.g., RHOA mRNA) may be complementary to a coding sequence of a RhoA transcript (e.g., having the nucleic acid sequence of SEQ ID NO: 14 or 15) and/or a non-coding sequence of a RhoA transcript (e.g., the 3’ untranslated region of a RhoA mRNA). In some embodiments, an inhibitory nucleic acid targets a RhoA transcript (e.g., RHOA mRNA) is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides of the nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48. In some embodiments, an inhibitory nucleic acid targets a RhoA transcript (e.g., RHOA mRNA) is complementary to the entirety of the nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48. Table 1. Exemplary inhibitory nucleic acid sequences for targeting a RHOA transcript *T’s may be substituted with U’s

Table 2. Exemplary inhibitory nucleic acid sequences for targeting a RHOA transcript

*T’s may be substituted with U’s

In some embodiments, an inhibitory nucleic acid (e.g., miRNA, amiRNA, siRNA, shRNA, or antisense oligonucleotide) that targets a RHOA transcript comprises one or more inhibitory nucleic acid sequences provided in Table 1. In some embodiments, an inhibitory nucleic acid (e.g., miRNA, amiRNA, siRNA, shRNA, or antisense oligonucleotide) that targets a RHOA transcript comprises or consists of the nucleic acid sequences of any one of SEQ ID NOs: 16-26. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises or consists of the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 17. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 18. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 20. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 22. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, an inhibitory nucleic acid that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 26.

In some embodiments, an inhibitory nucleic acid (e.g., miRNA, amiRNA, siRNA, shRNA, or antisense oligonucleotide) is complementary to a segment of a RhoA transcript (e.g., a RhoA mRNA). A segment of a nucleic acid (e.g., a RhoA transcript) may comprise or consist of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous bases of the nucleic acid. Thus, in some embodiments, an inhibitory nucleic acid that is complementary a segment of a RhoA transcript is complementary to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) continuous bases of a RhoA transcript. In some embodiments, an inhibitory nucleic acid (e.g., miRNA, amiRNA, siRNA, shRNA, or antisense oligonucleotide) is complementary to a segment of a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50.

In some embodiments, an inhibitory nucleic acid (e.g., miRNA, amiRNA, siRNA, shRNA, or antisense oligonucleotide) is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides of a nucleic acid sequence set forth in Table 2. In some embodiments, an inhibitory nucleic acid (e.g., miRNA, amiRNA, siRNA, shRNA, or antisense oligonucleotide) is complementary to the entirety of a nucleic acid sequence set forth in Table 2. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 38. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 39. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 40. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 41. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 42. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 43. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 44. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 45. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 46. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 47. In some embodiments, an inhibitory nucleic acid is complementary to at least 8, at least 10, at least 12, at least 15, or at least 20 contiguous nucleotides (or the entirety) of the nucleic acid sequence set forth in SEQ ID NO: 48.

In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequences of any one of SEQ ID NOs: 16-26. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, an amiRNA that targets a RHOA transcript comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, an amiRNA that targets a RHOA transcript comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 11.

A “microRNA” or “miRNA” is a small non-coding RNA molecule capable of mediating transcriptional or post-translational gene silencing. Typically, miRNA is transcribed as a hairpin or stem- loop (e.g., having a self-complementarity, single-stranded backbone) duplex structure, referred to as a primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into a pre-miRNA. The length of a pri-miRNA can vary. In some embodiments, a pri-miRNA ranges from about 100 to about 5000 base pairs (e.g., about 100, about 200, about 500, about 1000, about 1200, about 1500, about 1800, or about 2000 base pairs) in length. In some embodiments, a pri-miRNA is greater than 200 base pairs in length (e.g., 2500, 5000, 7000, 9000, or more base pairs in length).

Pre-miRNA, which is also characterized by a hairpin or stem-loop duplex structure, can also vary in length. In some embodiments, pre-miRNA ranges in size from about 40 base pairs in length to about 500 base pairs in length. In some embodiments, pre-miRNA ranges in size from about 50 to 100 base pairs in length. In some embodiments, pre-miRNA ranges in size from about 50 to about 90 base pairs in length (e.g., about 50, about 52, about 54, about 56, about 58, about 60, about 62, about 64, about 66, about 68, about 70, about 72, about 74, about 76, about 78, about 80, about 82, about 84, about 86, about 88, or about 90 base pairs in length).

Generally, pre-miRNA is exported into the cytoplasm, and enzymatically processed by Dicer to first produce an imperfect miRNA/miRNA* duplex and then a single-stranded mature miRNA molecule, which is subsequently loaded into the RNA-induced silencing complex (RISC). Typically, a mature miRNA molecule ranges in size from about 19 to about 30 base pairs in length. In some embodiments, a mature miRNA molecule is about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or 30 base pairs in length.

As used herein “artificial miRNA” or “amiRNA” refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA backbone, which is a precursor miRNA capable of producing a functional mature miRNA), in which the miRNA and miRNA* (e.g., passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/amiRNA* sequences that direct highly efficient RNA silencing of the targeted gene, for example as described by Eamens et al. (2014) Methods Mol. Biol. 1062:211-224. For example, in some embodiments, an artificial miRNA comprises a miR-155 pri-miRNA backbone into which a sequence encoding an inhibitory miRNA has been inserted in place of the endogenous miR-155 mature miRNA-encoding sequence. In some embodiments, miRNA (e.g., an amiRNA) as described by the disclosure comprises a miR-33 backbone sequence, a miR-155 backbone sequence, a miR-30 backbone sequence, a miR-30A backbone sequence, a miR-64 backbone sequence, or a miR-122 backbone sequence. In some embodiments, miRNA (e.g., an amiRNA) comprises a miR-33 backbone sequence. In some embodiments, an amiRNA comprises a miR- 33 scaffold, a miR-155 scaffold, a miR-30 scaffold, a miR-30A scaffold, a miR-64 scaffold, or a miR-122 scaffold. In some embodiments, an amiRNA comprises a miR-33 scaffold.

In some embodiments, an artificial microRNA is between 6-50 nucleotides in length. In some embodiments, an artificial microRNA is between 8-24 nucleotides in length. In some embodiments, an artificial microRNA is between 12-36 nucleotides in length. In some embodiments, an artificial microRNA is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

In some embodiments, an inhibitory nucleic acid sequence decreases expression of a target gene by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive). In some embodiments, an inhibitory nucleic acid sequence decreases expression of a target gene by between 75% and 90%. In some aspects, an inhibitory nucleic acid sequence decreases expression of a target gene by between 80% and 99%. In some embodiments, an inhibitory nucleic acid sequence targeting a RHOA transcript decreases expression of a RHOA transcript by between 50% and 99% (e.g., any integer between 50% and 99%, inclusive). In some embodiments, an inhibitory nucleic acid sequence targeting a RHOA transcript decreases expression of a RHOA transcript by between 75% and 90%. In some aspects, an inhibitory nucleic acid sequence targeting a RHOA transcript decreases expression of a RHOA transcript by between 80% and 99%.

As disclosed herein, “identity” of sequences refers to the measurement or calculation of the percent of identical matches between two or more sequences with gap alignments addressed by a mathematical model, algorithm, or computer program that is known to one of ordinary skill in the art. The percent identity of two sequences (e.g., nucleic acid or amino acid sequences) may, for example, be determined using Basic Local Alignment Search Tool (BLAST®) such as NBLAST® and XBLAST® programs (version 2.0). Alignment techniques such as Clustal Omega may be used for multiple sequence alignments. Other algorithms or alignment methods may include, but are not limited to, the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, or Fast Optimal Global Sequence Alignment Algorithm (FOGSAA). In some embodiments, a transgene may comprise one or more inhibitory nucleic acids (e.g., miRNAs or amiRNAs). In some embodiments, a transgene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more inhibitory nucleic acids (e.g., miRNAs or amiRNAs). It should be appreciated that in cases where a transgene encodes more than one inhibitory nucleic acids, each inhibitory nucleic acid may be positioned in any suitable location within the transgene. For example, a nucleic acid encoding a first amiRNA (e.g., a first artificial miRNA targeting RHOA) may be positioned 5’ to a nucleic acid sequence encoding a second miRNA (e.g., a second artificial miRNA targeting RHOA).

In some embodiments, a transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., a promoter, an enhancer, etc.). Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly-A) signals; and sequences that stabilize cytoplasmic mRNA. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissuespecific, are known in the art and may be utilized.

A "promoter" refers to a DNA sequence recognized by the machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned,", “operatively linked”, "under control" or "under transcriptional control" mean that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

As used herein, a nucleic acid sequence (e.g., a transgene encoding an inhibitory nucleic acid) and regulatory sequences are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a miRNA or an amiRNA, two DNA sequences are said to be operably linked if induction of a promoter in the 5’ regulatory sequence (e.g., 5’ untranslated region [5’ UTR]) results in the transcription of the miRNA or amiRNA, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a mutation, (2) interfere with the ability of the promoter region to direct the transcription of the miRNA or amiRNA, or (3) interfere with the ability of the corresponding inhibitory nucleic acid to knock down its intended target. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript effectively functions to knock down its target transcript. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more transcripts having been transcribed in frame.

Examples of constitutive promoters include, without limitation, the P-actin promoter (e.g., chicken P-actin [CB6] promoter), the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen] . In some embodiments, a promoter is an enhanced chicken P-actin promoter. In some embodiments, the chicken P-actin promoter comprises or consists of the sequence set forth in SEQ ID NO: 13.

In some embodiments, the regulatory sequences impart tissue- specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)); alpha-fetoprotein (AFP) promoter, (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal promoters such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503- 15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron- specific vgf gene promoter (Piccioli et al., Neuron, 15:373- 84 (1995)), among others which will be apparent to the skilled artisan.

In some embodiments, a tissue- specific promoter is an ocular tissue-specific promoter (e.g., trabecular meshwork- specific promoter). Examples of ocular tissue-specific promoters include, but are not limited to, promoters of, RPE65, rhodopsin, VMD2, opsin, etc. In some embodiments, a promoter is a trabecular meshwork- specific promoter. In some embodiments, a trabecular meshwork- specific promoter is a Chitinase-3-like 1 (Ch3Ll) promoter. In some embodiments, a Ch3Ll promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, a Ch3Ll promoter comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 12.

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline -repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

In some embodiments, an inducible promoter drives expression of a transgene under conditions of increased cell pressure (e.g., increased intraocular pressure). A pres sure- sensitive promoter, as used herein, refers to a promoter sequence that drives expression of a transgene in response to signaling associated with increased pressure exerted on a cell (e.g., signaling by Piezo channels, TRP channels, Rho GTPases (RhoA, Rael, Cdc42, etc.), YAP/TAZ, integrins, etc.). Non-limiting examples of pressure-sensitive (e.g., mechanosensitive) promoters are described in Acott et al., J Ocul Pharmacol Ther. 2014 Mar-Apr;30(2-3):94-101, the entirety of which is incorporated herein. Recombinant Adeno-Associated Viruses (rAAVs)

The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and/or delivered to a selected target cell. Recombinant AAV (rAAV) vectors are typically composed of, at a minimum: (i) a transgene and its regulatory sequences, and (ii) 5’ and 3’ AAV inverted terminal repeats (ITRs). The transgene may comprise, as disclosed elsewhere herein, one or more regions that encode one or more inhibitory nucleic acids (e.g., shRNA, miRNAs, amiRNAs, etc.) comprising a nucleic acid that targets an endogenous mRNA of a subject (e.g., a RHOA transcript). In some embodiments, an inhibitory nucleic acid is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue.

In some aspects, the instant disclosure provides a vector comprising a single, .s-acting wild-type ITR. In some embodiments, the ITR is a 5’ ITR. In some embodiments, the ITR is a 3’ ITR. Another example of such a molecule employed in the present disclosure is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' AAV ITR sequence and a 3’ hairpin-forming RNA sequence. Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5’ and 3’ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., the rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV ITR (e.g., an AAV2 ITR). In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125, and variants thereof. In some embodiments, the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS). The term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR). Without wishing to be bound by any particular theory, a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

As used herein, the term “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the AAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present. In general, scAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (WT) AAV TR at each end and a mutated TR (mTR) in the middle. The instant invention is based, in part, on the recognition that DNA fragments encoding RNA hairpin structures (e.g., shRNA, miRNA, and AmiRNA) can serve a function similar to a mutant inverted terminal repeat (mTR) during viral genome replication, generating self-complementary AAV vector genomes. For example, in some embodiments, the disclosure provides rAAV (e.g., self-complementary AAV; scAAV) vectors comprising a single- stranded self-complementary nucleic acid with inverted terminal repeats (ITRs) at each of two ends and a central portion comprising a promoter operably linked with a sequence encoding a hairpin-forming RNA (e.g., shRNA, miRNA, amiRNA, etc.). In some embodiments, the sequence encoding a hairpin-forming RNA (e.g., shRNA, miRNA, amiRNA, etc.) is substituted at a position of the self-complementary nucleic acid normally occupied by a mutant ITR.

In some embodiments, the rAAVs of the disclosure are pseudotyped rAAVs. For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX/Y (e.g., AAV2/1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue (e.g., ocular tissue).

Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner.

In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125. In some embodiments, an AAV capsid protein is of a serotype derived from a non-human primate, for example scAAVrh8, AAVrh39, or AAVrh43 serotype. In some embodiments, an AAV capsid protein is of an AAV2 serotype.

The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from HEK293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (z.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (z.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (z.e., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. In some embodiments, a host cell is a bacterial cell, yeast cell, insect cell (Sf9), or a mammalian (e.g., human, rodent, non-human primate, etc.) cell. In some embodiments, the mammalian cell is a HEK293 cell. In some embodiments, a mammalian cell is an ocular cell (e.g., a trabecular meshwork cell). A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced. In some aspects, the present disclosure provides a recombinant AAV comprising a capsid protein and an isolated nucleic acid comprising a first region encoding an AAV ITR and a second region comprising a transgene, wherein the transgene encodes an artificial microRNA. The artificial microRNA may decrease the expression of a target gene (e.g., RHOA) in a cell or tissue (e.g. ocular tissue) of a subject. In some embodiments, an rAAV comprises an artificial microRNA that decreases the expression of RhoA in a cell, a tissue, or a subject.

The foregoing methods for packaging recombinant vectors in desired AAV capsids to produce the rAAVs of the disclosure are not meant to be limiting and other suitable methods will be apparent to the skilled artisan.

Modes of Administration and Compositions

The isolated nucleic acids, nucleic acid sequences, compositions comprising nucleic acid sequences, vectors, rAAVs, and compositions of the present disclosure may be delivered to a subject in accordance with any appropriate methods known in the art. For example, a nucleic acid sequence, isolated nucleic acid, or rAAV may be preferably suspended in a physiologically compatible carrier (e.g., in a composition), and administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments a host animal does not include a human. In some embodiments, a subject is an adult. In some embodiments, a subject is a juvenile or infant.

In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a transgene encoding an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to any one of SEQ ID NOs: 16-26. In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a transgene encoding an amiRNA targeting a RHOA transcript, wherein the amiRNA comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to any one of SEQ ID NOs: 1-11. In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a transgene encoding an inhibitory nucleic acid having the sequence set forth in any one of SEQ ID NOs: 16-26 (or the complementary sequence thereof), or a portion thereof. In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a transgene encoding an amiRNA having the sequence set forth in any one of SEQ ID NOs: 1-11 (or the complementary sequence thereof), or a portion thereof

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 1.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 17. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 2.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 18. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 3.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 19. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 4.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 20. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 5.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 21. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 22. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 7.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 23. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 8.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 24. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 9.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 25. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 10.

In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an inhibitory nucleic acid targeting a RHOA transcript, wherein the inhibitory nucleic acid has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 26. In some embodiments, the nucleic acid sequence, isolated nucleic acid or rAAV comprises an amiRNA targeting a RHOA transcript, wherein the amiRNA has a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 11.

In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a promoter sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 12. In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a promoter sequence having the sequence set forth in SEQ ID NO: 12 (or the complementary sequence thereof), or a portion thereof.

In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a promoter sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to SEQ ID NO: 13. In some embodiments, the nucleic acid sequence, isolated nucleic acid, or rAAV comprises a promoter sequence having the sequence set forth in SEQ ID NO: 13 (or the complementary sequence thereof), or a portion thereof.

Delivery of the compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs to a mammalian subject may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in some embodiments, it may be desirable to deliver the virions directly to the ocular tissue of a subject. Recombinant AAVs may be delivered directly to the ocular tissue (e.g., the trabecular meshwork cells) by, e.g., intraocular injection with a needle, or using any suitable surgical techniques known in the art.

In some embodiments, compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs as described in the disclosure are administered by intracameral injection. The delivery procedures and methods can be any techniques that are known in the art and/or suitable for the present disclosure.

In some embodiments, compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs as described in the disclosure are administered by microneedle drug delivery such as transdermal application. In some embodiments, compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs as described in the disclosure are administered by the use of dermal patches for providing controlled delivery. A dermal patch, skin patch, or the like as used herein refers to a medicated adhesive patch that is placed on the skin to deliver a specific dose of a composition into the skin. Dermal or skin patches can include but are not limited to single-layer drug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, and vapor patches. Alternatively, or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel. In some embodiments, permeation enhancers can be used for enhancing the permeation of compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs in the patch.

Aspects of the instant disclosure relate to compositions comprising a recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more artificial miRNAs, for example. In some embodiments, the nucleic acid further comprises one or more AAV ITRs. In some embodiments, a composition further comprises a pharmaceutically acceptable carrier. In some embodiments, compositions comprise a recombinant AAV comprising a capsid protein and a nucleic acid comprising a first region encoding an AAV ITR and a second region comprising a transgene, wherein the transgene encodes at least one artificial microRNA that targets RhoA.

The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.

Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the composition, nucleic acid sequence, isolated nucleic acid, or rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intracameral injection into the eye), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.

The dose of compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.

In some embodiments, an “effective amount” of a substance is an amount sufficient to produce a desired effect (e.g., to transduce ocular (e.g., trabecular meshwork) cells). In some embodiments, an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or infect in the context of rAAV-mediated delivery) a sufficient number of target cells of a target tissue of a subject. In some embodiments, a target tissue is ocular tissue. In some embodiments, an effective amount of an isolated nucleic acid (e.g., which may be delivered via an rAAV) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to reduce intraocular pressure, prevent blindness, etc. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue as described elsewhere in the disclosure. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 10⁹ to 10¹⁶ genome copies. In some cases, a dosage between about 10¹¹ to 10¹³ rAAV genome copies is appropriate. In certain embodiments, 10¹² or 10¹³ rAAV genome copies are effective to target ocular tissue.

In some embodiments, a dose of compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose is administered to a subject no more than bi-weekly (e.g., once in a two calendar-week period). In some embodiments, a dose is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose is administered to a subject no more than once per six calendar months. In some embodiments, a dose is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~10¹³ GC/ml or more). Methods for reducing aggregation of rAAVs are well known in the art and include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.) In some embodiments, administering an isolated nucleic acid, an rAAV, and/or a vector as disclosed herein results in a decrease of a RhoA protein (e.g., a mouse or a human RhoA protein) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administering an isolated nucleic acid, an rAAV, and/or a vector as disclosed herein results in a decrease of a RhoA protein (e.g., a mouse or a human RhoA protein) by about 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 85- 90%, 90-95%, 95-100%, 1-100%, 1-20%, 20-40%, 40-60%, 60-80%, or 80-100% compared to a control.

In some embodiments, administering an isolated nucleic acid, an rAAV, and/or a vector as disclosed herein results in a decrease of a RHOA transcript (e.g., a mouse or a human RHOA transcript) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administering an isolated nucleic acid, an rAAV, and/or a vector as disclosed herein results in a decrease of a RHOA transcript (e.g., a mouse or a human RHOA transcript) by about 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70- 75%, 75-80%, 85-90%, 90-95%, 95-100%, 1-100%, 1-20%, 20-40%, 40-60%, 60-80%, or 80- 100% compared to a control.

Generally, intraocular pressure (IOP) is measured in millimeters of mercury (mmHg) and is normally in the range of about 10 mmHg to about 21 mmHg in healthy subjects. In some embodiments, administering a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein ameliorates (e.g., reduces) intraocular pressure in a subject with increased ocular pressure (e.g., a subject with glaucoma) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administration of a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein results in decreased intraocular pressure (e.g., decreased intraocular pressure by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times) in a subject with increased intraocular pressure (e.g., a subject with glaucoma) relative to the subject prior to administration.

In some embodiments, administering a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein ameliorates (e.g., reduces) RhoA levels (e.g., RHOA transcript or RhoA protein) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administration of a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein results in decreased RhoA levels (e.g., RHOA transcript or RhoA protein) (e.g., decreased RhoA levels (e.g., RHOA transcript or RhoA protein) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times) in a subject relative to the subject prior to administration.

In some embodiments, administering a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein reduces ROCK1/2 signaling by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administration of a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein results in reduced ROCK1/2 signaling (e.g., decreased ROCK1/2 signaling by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times) in a subject relative to the subject prior to administration.

In some embodiments, administering a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein increases aqueous humor outflow by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5- fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, administration of a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein results in increased aqueous humor outflow (e.g., increased aqueous humor outflow by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times) in a subject relative to the subject prior to administration.

In some embodiments, administering a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein reduces trabecular meshwork contraction by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000- fold compared to a control. In some embodiments, administration of a composition, a nucleic acid sequence, an isolated nucleic acid, and/or an rAAV as disclosed herein results in reduced trabecular meshwork contraction (e.g., decreased trabecular meshwork contraction by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times) in a subject relative to the subject prior to administration.

As used herein, improvement or amelioration is relative to a control. The control can be in a state that is prior to the administration of the composition, isolated nucleic acid, rAAV, and/or vector. In some embodiments, the improvement or amelioration is relative to a subject that has not been administered the composition, nucleic acid sequence, isolated nucleic acid, and/or rAAV.

In some embodiments, a “control” can refer to a subject that has increased (e.g., above average) intraocular pressure while not being treated by the methods and compositions described in the present disclosure or by any other methods. In some embodiments, a “control” can refer to a subject that has glaucoma while not being treated by the methods and compositions described in the present disclosure or by any other methods.

Formulation of pharmaceutically acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either intraocularly, subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, intramuscularly, intradermally, intrathecally, femoral intramedullary, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used for delivery. In some embodiments, a preferred mode of administration is by intraocular injection (e.g., intracameral injection).

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the compositions, nucleic acid sequences, isolated nucleic acids, or rAAVs may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the compositions to a host. Sonophoresis (z.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).

Exemplary embodiments of the invention will be described in more detail by the following examples. These embodiments are exemplary of the invention, which one skilled in the art will recognize is not limited to the exemplary embodiments.

EXAMPLES

Example 1: Screening of artificial microRNAs targeting RhoA in vitro

Eleven unique amiR scaffolds were designed to target either the coding sequence (CDS) or the 3’ untranslated region (3’ UTR) of mouse and human RhoA transcripts (mRhoA [SEQ ID NO: 49] and hsRhoA [SEQ ID NO: 50], respectively) (FIG. 1A). Each scaffold was inserted into a plasmid containing an Egfp transgene under the control of the ubiquitous CB6 promoter. For each plasmid, the amiR scaffolds were inserted into an intron 5’ to the Egfp coding sequence (SEQ ID NOs: 27-37). To test the efficacy of RhoA knockdown for each scaffold, luciferase assays were performed using a PsiCheck reporter system engineered to express RhoA (Promega Corporation. PsiCHECK™-l and PsiCHECK™-2 Vectors. Promega Corporation website. https://www.promega.com/products/rna-analysis/ma-interference/psicheck-l-and-psicheck-2- vectors/?catNum=C8021. Accessed March 27, 2024). Plasmid constructs expressing the unique amiR scaffolds were co-transfected with the psiCheck vector into HEK293 and human trabecular meshwork (TM1) cells at ratios of 1:1 or 0.5:1 amiR-RhoA-EGFP:psiCheck, and luciferase activity was read 48 hours later (FIG. IB). Quantitation of relative luciferase activity demonstrated that most RhoA-targeting amiR designs successfully knocked down mouse and human rhoa transcript expression in both HEK293 and TM1 cells (FIGs. 1C and ID), with amiR4, amiRlO, and amiRl 1 showing the highest knockdown efficiency.

To achieve specificity of expression in trabecular meshwork cells, plasmids were engineered to express EGFP, amiR4, amiRlO, or amiRl 1 under the control of a Chitinase-3- likel (Ch3Ll) promoter (SEQ ID NO: 12), which is specific to trabecular meshwork cells (Liton et al., Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter. Invest Ophthalmol Vis Sci. 2005 Jan;46(l): 183-90). Plasmids expressing EGFP under the control of the Ch3Ll or ubiquitous CB6 promoter (SEQ ID NO: 13) (Ch3Ll-EGFP and CB6-EGFP, respectively) were tested for expression in TM1 cells, and Neuro-2A cells were used as controls. In contrast to the CB6-EGFP construct, which showed expression in both the TM1 and Neuro-2A cells, the expression of Ch3-L1EGFP was specific to TM1 cells (FIG. 2A). The plasmids expressing amiR4, amiRlO, and amiRl 1 under the control of the Ch3Ll promoter (Ch3Ll-amiR4, Ch3Ll-amiR10, and Ch3Ll-amiRl l, respectively) were then tested for their ability to knock down RhoA in TM1 cells using the psiCheck luciferase reporter system as described above. Quantitation of the relative luciferase activity demonstrated that all three amiR designs efficiently knocked down both human and mouse rhoa transcripts when driven by the Ch3Ll promoter (FIG. 2B).

To confirm that the knockdown of rhoa transcript using Ch3Ll-amiR4, Ch3Ll-amiR10, and Ch3Ll-amiRl 1 translated to a decrease in RhoA protein, the amiR candidates were transiently transfected in TM1 cells, and the RhoA protein levels assessed 48 hours later. All three amiR designs resulted in efficient knockdown of RhoA protein (FIG. 3). amiR scaffolds are processed by the microRNA (miRNA) processing machinery in cells (Xie et al., Effective and Accurate Gene Silencing by a Recombinant AAV-Compatible MicroRNA Scaffold. Mol Ther. 2020 Feb 5;28(2):422-430). The processing pathway consists of a self-complementary double-stranded RNA that is cleaved by Drosha and Dicer nucleases to generate mature single- stranded miRNAs, which then later become part of the RNA-induced silencing complex (RISC) (O’Brien et al., Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front Endocrinol (Lausanne). 2018 Aug 3;9:402). A key step in the maturation process of miRNA is to preserve the intended single strand, known as the guide strand, which is complementary to the target transcript. The other strand, known as the passenger strand, is degraded. RISC then binds to the target transcripts and degrades them, resulting in post-transcriptional regulation of gene expression. Thus, an increased guide-to- passenger ratio is indicative of effective target knockdown, and a decrease can cause off-target effects.

To assess the precision of miRNA end-processing and to evaluate off-target effects, TM1 cells were transduced with AAV2-packaged amiR constructs, and small RNA sequencing was performed. The major objects of this evaluation included the determination of: 1) the guide-to- passenger strand ratios as a measure of efficient amiR design; 2) the 5’ and 3’ end processing to evaluate the precision of seed-sequence maturation; and 3) the expression of endogenous miRNAs to evaluate possible off-target effects. Quantitation of guide-to-passenger strand ratios (G/P ratio) demonstrated amiR4 to have the best G/P ratio, followed by amiRl 1, and finally amiRlO (FIG. 4A), though all G/P ratios were >1, indicating efficient amiR design. Analysis of the 5’- and 3’-end processing of mature miRNAs showed more than 90% accuracy for all three amiR candidates (FIG. 4B). Finally, expression levels of endogenous miRNAs were largely unaffected following transduction, with only a handful showing overexpression in the presence of amiR4, amiRlO, and amiRl 1 (FIG. 4C).

Example 2: amiR knockdown of RhoA in vivo

In primary open angle glaucoma (POAG), one of the high-risk factors that aggravates blindness of the accumulation of excess aqueous humor (AH). This accumulation of AH results in increased intraocular pressure (IOP) and causes damage to the optic nerve, leading to loss of sight (FIG. 5A). Maintenance of an optimum IOP is an intricate process requiring a balance between AH secretion by the cells of the ciliary body and its drainage through the cellular layers of the trabecular meshwork and Schlemm’ s canal (FIG. 5B). A major hindrance to the outflow of AH is increased contraction of the trabecular meshwork layers, which is controlled in part by the phosphorylation of substrates (e.g., myosin light chain (MLC), myosin phosphatase target subunit 1, LIM kinases 1/2) by Rho-associated serine-threonine kinases (ROCK1/2). RhoA is a ubiquitously expressed small GTPase that, when activated (i.e., when bound to GTP), can associate with and activate ROCK1/2 (FIG. 6), leading to downstream actin polymerization and cellular contraction.

To test whether delivering amiRs targeting RhoA directly to the eyes could affect IOP, amiRlO expression cassettes were packaged into AAV2 capsids (AAV2-CB6-amiR10) and injected into mouse eyes by intracameral injection at four different doses (1.67E8, 3.33E8, 7.35E8, and 1.47E9 vg/eye). Three weeks later, the mice injected with 7.35E8 and 1.47E9 vg/eye showed a significant reduction in IOP compared to mice injected with control vector (AAV2-CB6-Egfp) (FIG. 7), indicating AAV-based gene therapy targeting RhoA to be a promising therapeutic method of treating subjects with increased IOP (e.g., with glaucoma).

ADDITIONAL SEQUENCES

>Ch3Ll Promoter (SEQ ID NO: 12)

TTAAGCCTGCAAAGAATGGAGTTGTCCTGGATATTTGGCCAAAAAAAAAATGTATC

CACAAACAGGGACGTAATCAGGCAGGGAGCCTCGTTAAGAAGTTTTGTTCTTGTCC

TAGGAGTGATGAGAGATCACTGAAGGATTTAGAGAGGGGCTGTATCATCAGGCTTG

GGTTCCAAAGCCTCACTGAGAGAGTTGGGGAGCTGACTGATGTCAGATGCTCGTGC

AGCCGCCCCGTAGGGCCTGTATTTCCTCCATGGTGCCTCACTGCAGCACCGAGCTTG

CAAAAGATCCTCTCTCTTTATGGGAATTTCAAAACAGAAGCAAAATAGCACCGGGG

CTTAAAGCATTCTTGGGAATTTCCCTGTCTTTCCCTCTAAATAATCAGCATGTAAATT

GCAAAAAAAAAAAAAAAAAAAAAAAAAAAGACACGGGCCCAAAAGGGAGCGCTC

AGTTTCAGGCTCTTTGCTTTCCTTCCTCCCGAGGCTCTCTGGCCCTTACCCAGCCTGA

AGACAGAAAGTGTGAGGGGGAGGGTAGGAAGGTAGGTCAAGCAGGGCAATGCTGA

GCCTGGGAAGAAAACAACAGCCTTGTTTAGGGCACTGTGGCTTACGTAACTAAATT

GTGCCCAGTTTCCACCTGGCCAGGGGCCTGGAGTGAATGCTGAAGATGCAAAGGTA

GAGGCTGCCAGAAAAGCCAGGAAATTGCTGGCAAGAAAGGCCAGTGGTGGGGTGC

AGGAGTGGGAGGAAGGCTGGGAAATGCGGCTGAGTCACATCTCCAGAAGCCCCCC

ATCATCACCCTAGTGGCTCTTCTGCTGGCAGGTGCCTCATGAAGACCTGACCCAAAG

TTTTCAAAACTCTGCGGTTTCTCAACCCTCCTCTGGTAATCCATAGTACTCCCCCGCC

TCCACTTGCCAGCCTCGTGATTCCTTCATTGACACATAGCTCAGTTCCCATAAAAGG

GCTGGTTTGCCGCGTCGGGGAGTGGAGTGGGACAGGTATATAAAGGAAGTACAGG

GCCTGGGGAAGAGGCCCTGTCTAGGTAGCTGGCACCAGGAGCCGTGGG

>CB6 Promoter (SEQ ID NO: 13)

TCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTAT

TTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGG

CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCA

ATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGG

CCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATC

>Mouse RhoA mRNA, coding sequence (SEQ ID NO: 14)

ATGGCTGCCATCAGGAAGAAACTGGTGATTGTTGGTGATGGAGCTTGTGGTAAGAC ATGCTTGCTCATAGTCTTCAGCAAGGACCAGTTCCCAGAGGTCTATGTGCCCACGGT GTTTGAAAACTATGTGGCGGATATCGAGGTGGATGGGAAGCAGGTAGAGTTGGCTT TATGGGACACAGCTGGACAGGAAGATTATGACCGCCTGCGGCCTCTCTCTTATCCA

GACACCGATGTTATATTGATGTGTTTTTCCATTGACAGCCCTGATAGTTTAGAAAAC

ATCCCAGAAAAATGGACTCCAGAAGTCAAGCATTTCTGTCCAAATGTGCCCATCAT CCTGGTTGGGAACAAGAAGGACCTTCGGAATGACGAGCACACGAGACGGGAGTTG GCCAAAATGAAGCAGGAGCCGGTAAAACCTGAAGAAGGCAGAGATATGGCAAACA GGATTGGCGCTTTTGGGTACATGGAGTGTTCAGCAAAGACCAAAGATGGAGTGAGA

GAGGTTTTTGAGATGGCCACGAGAGCTGCTCTGCAAGCTAGACGTGGGAAGAAAAA GTCTGGGTGCCTCATCTTGTGA >Human RhoA mRNA, coding sequence (SEQ ID NO: 15)

ATGGCTGCCATCCGGAAGAAACTGGTGATTGTTGGTGATGGAGCCTGTGGAAAGAC

ATGCTTGCTCATAGTCTTCAGCAAGGACCAGTTCCCAGAGGTGTATGTGCCCACAGT

GTTTGAGAACTATGTGGCAGATATCGAGGTGGATGGAAAGCAGGTAGAGTTGGCTT

TGTGGGACACAGCTGGGCAGGAAGATTATGATCGCCTGAGGCCCCTCTCCTACCCA

GATACCGATGTTATACTGATGTGTTTTTCCATCGACAGCCCTGATAGTTTAGAAAAC

ATCCCAGAAAAGTGGACCCCAGAAGTCAAGCATTTCTGTCCCAACGTGCCCATCAT

CCTGGTTGGGAATAAGAAGGATCTTCGGAATGATGAGCACACAAGGCGGGAGCTA

GCCAAGATGAAGCAGGAGCCGGTGAAACCTGAAGAAGGCAGAGATATGGCAAACA

GGATTGGCGCTTTTGGGTACATGGAGTGTTCAGCAAAGACCAAAGATGGAGTGAGA GAGGTTTTTGAAATGGCTACGAGAGCTGCTCTGCAAGCTAGACGTGGGAAGAAAAA ATCTGGGTGCCTTGTCTTGTGA

>Mouse RhoA mRNA, full transcript, NCBI Reference Sequence: NM_001313962.1 (SEQ ID

NO: 49)

GGGCCTGGTGGAGCCCTAGCCGCCCGCCCTCCTCGTCGCTTCCTCTCAGGGTTGCGC

CGGGCTGGGCGGGGGCTCCCGGCCGGTCCGCGACCGCGCTGGGGTAGCCTGCCACT

GCGAAGCGCGTGTGGCTGTCGGGAGTTGGACTAGCCTTGCATCTGAGAAGTTCCAG

GTACTTTGTACAACTGCATCCCAGAACCTGTGTGTTTTCAGCACCTTTATAAGTGAT

GGCTGCCATCAGGAAGAAACTGGTGATTGTTGGTGATGGAGCTTGTGGTAAGACAT

GCTTGCTCATAGTCTTCAGCAAGGACCAGTTCCCAGAGGTCTATGTGCCCACGGTGT

TTGAAAACTATGTGGCGGATATCGAGGTGGATGGGAAGCAGGTAGAGTTGGCTTTA

TGGGACACAGCTGGACAGGAAGATTATGACCGCCTGCGGCCTCTCTCTTATCCAGA

CACCGATGTTATATTGATGTGTTTTTCCATTGACAGCCCTGATAGTTTAGAAAACAT CCCAGAAAAATGGACTCCAGAAGTCAAGCATTTCTGTCCAAATGTGCCCATCATCCT GGTTGGGAACAAGAAGGACCTTCGGAATGACGAGCACACGAGACGGGAGTTGGCC

AAAATGAAGCAGGAGCCGGTAAAACCTGAAGAAGGCAGAGATATGGCAAACAGGA

T _GT_TG_TTG_TC_TG_GC_AT_GTT_AT_TG_GG_GG_CT_CA_AC_CA_GT_AG_GG_AA_GG_CT_TG_GT_CT_TC_CA_TG_GC_CA_AA_AA_GG_CA_TACC_GA_AA_CA_GTG_GA_GT_GG_AG_AA_GG_ATG_AA_AG_AA_AG_GA_TCG

TGGGTGCCTCATCTTGTGAAGCCTTGTGAACGCAGCCTCATGCGGTTAATTTGAAGT

GCTGTTTATTAATCTTAGTGTATGATTACTGGCCTTTTCATTTATCTATAATTTACCT

AAGATTACAAATCAGAAGTCATCTTGCTACCAGTATTTAGAAGCCAACCACGATTA TTAATAATGTCCAACCTGTCTGACCAGCCAGGGTCCTTCTGACACTGCTCTAACAGC CCTCTCTGCACTCCACCTGACACCAGGCGCTAATTCAAAGAATTTCTTAACTTCTTG

CTTCTTTCTAGAAAGAGAAACAGTTGGTAACTTTTGTGAATTAGGCTGTAACTACTT

TATAACTAACATGTCCTGCCTACTTTCTGTCAACTGCAAGAACTCTGGTGAGTCACT

ACTTCAGAGCTTTCCTTGTTAACAGACTCCATTGCCAGAGCTCTGGGGTGGGTATTC

AGTTTTTTGAAATCTTGCTCAGCCAGAAAGGCCCAAGTCCACGCAGCTGTTGCAGA GTTACAGTTCTGTGGTCTCATGTTAGTTACCTTATAGTTACTGTGTAATTAGTGCCAC TTAATGTATGTTACCAAAAAATAAATATATCTACCCCAGACTAGATGTAGTATTTTT

TGTATAATTGGATTTCCTAATACTGATATTTGTCATTCCCACAGAAAGTGTATTGCTT

CTTTTCTTTTTCTTTCTTTCTTTCTTTTTTTCTTTTTTTTTTTTTTTTTTAAGAAAGTGTA

TTTGGAAATAAAGTCAGATGGAAGAATTCATTTTTAAAATTCCCATTTTGTCACTTT

CTCTGATAAAATATGGCCATATCCCTTATTCAGCCCTATATATCATTCTAGTACCCCT

TTCCAGACTGGACTAAGTAGGAATTTGGTTTCCCGCCTGAGGCAGTTATACTTTGGA

GGTGGCATAGCCTTTCTCACCTGGACTGCAGGGTCTGGCTCTAAGTCACAGTGCTCC TTTCTCCACACTGTATCCAAGTTGCCTCCCAGAGGAGCCACCAGTTCTTATGGGTGG CAGTGGGTGGGGGTGGGGGGGTCTCTTCTCTCCAGCTGACTAAACATTTTTCTGTAC CAGTTAATTTTTCCAACTAATAGAATAAAGGCAGTTTTCTAAATTTCCTGTAAAAAA AAAAAAAAAAA

>Human RhoA mRNA, full transcript, NCBI Reference Sequence: NM_001664.4 (SEQ ID NO:

50)

GCTCTCTCGCGCTACCCTCCCGCCGCCCGCGGTCCTCCGTCGGTTCTCTCGTTAGTCC

ACGGTCTGGTCTTCAGCTACCCGCCTTCGTCTCCGAGTTTGCGACTCGCGGACCGGC

GTCCCCGGCGCGAAGAGGCTGGACTCGGATTCGTTGCCTGAGCAATGGCTGCCATC

CGGAAGAAACTGGTGATTGTTGGTGATGGAGCCTGTGGAAAGACATGCTTGCTCAT

AGTCTTCAGCAAGGACCAGTTCCCAGAGGTGTATGTGCCCACAGTGTTTGAGAACT

ATGTGGCAGATATCGAGGTGGATGGAAAGCAGGTAGAGTTGGCTTTGTGGGACACA

GCTGGGCAGGAAGATTATGATCGCCTGAGGCCCCTCTCCTACCCAGATACCGATGTT

ATACTGATGTGTTTTTCCATCGACAGCCCTGATAGTTTAGAAAACATCCCAGAAAAG

TGGACCCCAGAAGTCAAGCATTTCTGTCCCAACGTGCCCATCATCCTGGTTGGGAAT

AAGAAGGATCTTCGGAATGATGAGCACACAAGGCGGGAGCTAGCCAAGATGAAGC

AGGAGCCGGTGAAACCTGAAGAAGGCAGAGATATGGCAAACAGGATTGGCGCTTT

TGGGTACATGGAGTGTTCAGCAAAGACCAAAGATGGAGTGAGAGAGGTTTTTGAAA

TGGCTACGAGAGCTGCTCTGCAAGCTAGACGTGGGAAGAAAAAATCTGGGTGCCTT

GTCTTGTGAAACCTTGCTGCAAGCACAGCCCTTATGCGGTTAATTTTGAAGTGCTGT

TTATTAATCTTAGTGTATGATTACTGGCCTTTTTCATTTATCTATAATTTACCTAAGA

TTACAAATCAGAAGTCATCTTGCTACCAGTATTTAGAAGCCAACTATGATTATTAAC

GATGTCCAACCCGTCTGGCCCACCAGGGTCCTTTTGACACTGCTCTAACAGCCCTCC

TCTGCACTCCCACCTGACACACCAGGCGCTAATTCAAGGAATTTCTTAACTTCTTGC

TTCTTTCTAGAAAGAGAAACAGTTGGTAACTTTTGTGAATTAGGCTGTAACTACTTT

ATAACTAACATGTCCTGCCTATTATCTGTCAGCTGCAAGGTACTCTGGTGAGTCACC

ACTTCAGGGCTTTACTCCGTAACAGATTTTGTTGGCATAGCTCTGGGGTGGGCAGTT

TTTTGAAAATGGGCTCAACCAGAAAAGCCCAAGTTCATGCAGCTGTGGCAGAGTTA

CAGTTCTGTGGTTTCATGTTAGTTACCTTATAGTTACTGTGTAATTAGTGCCACTTAA

TGTATGTTACCAAAAATAAATATATCTACCCCAGACTAGATGTAGTATTTTTTGTAT

AATTGGATTTCCTAATACTGTCATCCTCAAAGAAAGTGTATTGGTTTTTTAAAAAAG

AAAGTGTATTTGGAAATAAAGTCAGATGGAAAATTCATTTTTTAAATTCCCGTTTTG

TCACTTTTTCTGATAAAAGATGGCCATATTACCCCTTTTCGGCCCCATGTATCTCAGT

ACCCCATGGAGCTGGGCTAAGTAAATAGGAATTGGTTTCACGCCTGAGGCAATTAG

ACACTTTGGAAGATGGCATAACCTGTCTCACCTGGACTTAAGCATCTGGCTCTAATT

CACAGTGCTCTTTTCTCCTCACTGTATCCAGGTTCCCTCCCAGAGGAGCCACCAGTT

CTCATGGGTGGCACTCAGTCTCTCTTCTCTCCAGCTGACTAAACTTTTTTTCTGTACC AGTTAATTTTTCCAACTACTAATAGAATAAAGGCAGTTTTCTAAA

>amiRl-RhoA-EGFP (SEQ ID NO: 27)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGCAACAATCACCAGTTTCT

TCCTGTTCTGGCAATACCTGGGAAGAAAGAGATGATTGTTGCACGGAGGCCTGCCC

TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC

CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC

AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA

CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG

ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC

CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC

CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC

CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT

CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA

GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC

CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG

GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG

AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC

TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG

GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT

TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG

GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC

TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA

TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR2-RhoA-EGFP (SEQ ID NO: 28)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTCTCACTCCATCTTTGGT

CTTTGTTCTGGCAATACCTGAAGACCAATCACGGAGTGAGACACGGAGGCCTGCCC

TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC

CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC

AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA

CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG

ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC

CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC

CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC

CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT

CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA

GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC

CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG

GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG

AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC

TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG

GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT

TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG

GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC

TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA

TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR3-RhoA-EGFP (SEQ ID NO: 29)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTCTCTCACTCCATCTTTG GTCTGTTCTGGCAATACCTGGACCAAAGTAGAAGTGAGAGACACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR4-RhoA-EGFP (SEQ ID NO: 30)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTATCTCTGCCTTCTTCAG

GTTTGTTCTGGCAATACCTGAACCTGAACTAAGCAGAGATACACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC

CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC

AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA

CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG

ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC

CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC

CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC

CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT

CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA

GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC

CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG

GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG

AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC

TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG

GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT

TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG

GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC

TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA

TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR5-RhoA-EGFP (SEQ ID NO: 31)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTCCATCACCAACAATCAC

CAGTGTTCTGGCAATACCTGCTGGTGATACTCGGTGATGGACACGGAGGCCTGCCCT

GACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTACC

TAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATCA

AAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTAC TTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATG

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA

CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC

ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC

CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC

CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG

AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA

GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC

GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG

CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC

CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC

GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGT

GACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCATC

AAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAG

TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC

ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG

TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA

AGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCC

TAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC

GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA

GCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGG

GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGC

TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT

GAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG

TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT

TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG

GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGA

TTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTT

GACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT

CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT

TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR6-RhoA-EGFP (SEQ ID NO: 32)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTTCCCAACCAGGATGAT

GGGCTGTTCTGGCAATACCTGGCCCATCAAGCCGGTTGGGAACACGGAGGCCTGCC

CTGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTA

CCTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAAT

CAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTT

ACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCA

TGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG

GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG

CCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC

CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT

CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG

TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTC

AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA

ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATC

CGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC

CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGT

CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC

GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCA

TCAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCT

AGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGT

GCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGT

AGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG

GGAAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAAC

CCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG

GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG

CGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGAC

TGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCC

AGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAG

CCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG

TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCG

CTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCG

GGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT

TGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCC

TTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAAC

ACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCC

TATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT

ATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATT

TGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGA

TAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC

GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC

TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA

CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCA

ATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC

GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA

CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA

GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC

GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCG

CCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA

CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTA

CTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGC

AGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG

AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC

CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA

AATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA

CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGG

ATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTT T _TC_TTG_TT_TT_TC_CC_TA_GC_CT_GG_CA_GG_TC_AG_AT_TC_CA_TG_GA_CC_TC_GC_CC_TG_TT_GA_CG_AA_AA_AA_CA_AG_AA_AT_AC_AA_AA_AA_CG_CG_AA_CT_CC_GT_CT_TC_AT_CT_CG_AA_GG_CA_GT_GC_TC_GT

GTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC

AGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC

AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT

GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC

GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGG

AGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC

ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT

GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG

CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGC

TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTA

TTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGC

GAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCG

CGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCG

GGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC

TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT

TCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATT

AGG

>amiR7-RhoA-EGFP (SEQ ID NO: 33)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGCATATCTCTGCCTTCTTC AGGTGTTCTGGCAATACCTGCCTGAAGATCGTAGAGATATGCACGGAGGCCTGCCC TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC

CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC

CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT

CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA

GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC

CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG

GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG

AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC

TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG

GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT

TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG

GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC

TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA

TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR8-RhoA-EGFP (SEQ ID NO: 34)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTTTTCTGGGATGTTTTCT

AAATGTTCTGGCAATACCTGTTTAGAAATGACCCCAGAAAACACGGAGGCCTGCCC

TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC

CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC

AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA

CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG

ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC

CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC

CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC

CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT

CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA

GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC

CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG

GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG

AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC

TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG

GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT

TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG

GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC

TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA

TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR10-RhoA-EGFP (SEQ ID NO: 35)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGTCCATCTGACTTTATTTC

CAATGTTCTGGCAATACCTGTTGGAAATTTAATCAGATGGACACGGAGGCCTGCCCT

GACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTACC

TAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATCA

AAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTAC

TTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATG

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA

CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC

ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC

CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC

CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG

AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA

GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC

GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG

CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGT GACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCATC AAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAG TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCC TAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA GCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGG GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGC TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT GAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGA TTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTT GACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG

AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiRll-RhoA-EGFP (SEQ ID NO: 36)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGAAGGCCAGTAATCATAC

ACTATGTTCTGGCAATACCTGTAGTGTATCTTCACTGGCCTTCACGGAGGCCTGCCC

TGACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTAC

CTAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATC

AAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTA

CTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCAT

GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG

ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC

CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC

CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC

CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT

GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCC

GCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC

CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC

CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCAT CAAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTA

GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC

CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG

GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG

AGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC

TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG

GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT

TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG

GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT

TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC

TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA

TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC

CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG

GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA

CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT

ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG

ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC

CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT

CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA

AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG

TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT

GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC

GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG

GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA

CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

>amiR12-RhoA-EGFP (SEQ ID NO: 37)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCAT

GCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACATTGATTATTGACTCTGG

TCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCGCCC

ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG

ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCG

AGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGG

GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG

AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGG

GATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAG

TTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCAGATCTAGGGCTCTG

CGTTTGCTCCAGGTAGTCCGCTGCTCCCTTGGGCCTGGGCCCACTGACAGCCCTGGT

GCCTCTGGCCGGCTGCACACCTCCTGGCGGGCAGCTGTGAGTAGTTACAGCCTAATT

CACTGTTCTGGCAATACCTGGTGAATTACCCCGTAACTACTCACGGAGGCCTGCCCT

GACTGCCCACGGTGCCGTGGCCAAAGAGGATCTAAGGGCACCGCTGAGGGCCTACC

TAACCATCGTGGGGAATAAGGACAGTGTCACCCGGATCCGGTGGTGGTGCAAATCA

AAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTAC

TTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATG

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA

CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC

ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC

CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC

CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG

AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA

GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC

GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG

CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC

CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC

GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGT

GACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCATC

AAGCTTATCGATACCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAG

TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC

ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCC TAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA GCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGG GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGC TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT GAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGA TTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTT GACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA

TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG

TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA

GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG

Claims

CLAIMS What is claimed is:

1. An isolated nucleic acid comprising a transgene encoding an inhibitory nucleic acid targeting a RhoA messenger RNA (mRNA), wherein the transgene is flanked by adeno- associated virus (AAV) inverted terminal repeats (ITRs).

2. The isolated nucleic acid of claim 1, wherein the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26.

3. An isolated nucleic acid comprising an inhibitory nucleic acid having a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26.

4. The isolated nucleic acid of any one of claims 1-3, wherein the inhibitory nucleic acid is an artificial microRNA (amiRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an antisense oligonucleotide (ASO).

5. The isolated nucleic acid of any one of claims 1-4, wherein the inhibitory nucleic acid is an artificial microRNA (amiRNA).

6. The isolated nucleic acid of claim 4 or 5, wherein the amiRNA comprises a miRNA backbone sequence selected from a miR-33, a miR-155, a miR-30, a miR-30A, a miR-64, or a miR-122 backbone sequence.

7. The isolated nucleic acid of any one of claims 1-6, wherein the inhibitory nucleic acid is an amiRNA comprising a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-11.

8. The isolated nucleic acid of any one of claims 1-7, wherein the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, or 99% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 19, 24, or 25.

9. The isolated nucleic acid of claim 8, wherein the inhibitory nucleic acid is an amiRNA comprising a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 9, or 10.

10. The isolated nucleic acid of any one of claims 1-9, wherein the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50.

11. The isolated nucleic acid of any one of claims 1-10, wherein the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

12. The isolated nucleic acid of any one of claims 1-11, wherein the inhibitory nucleic acid is complementary to a segment of a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50.

13. The isolated nucleic acid of claim 12, wherein the segment of nucleic acid comprises 2- 24 continuous nucleotides.

14. The isolated nucleic acid of any one of claims 1-13, wherein the inhibitory nucleic acid is complementary to a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

15. The isolated nucleic acid of any one of claims 1-14, wherein the nucleic acid encoding the inhibitory nucleic acid is operably linked to a promoter.

16. The isolated nucleic acid of claim 15, wherein the promoter comprises a trabecular meshwork-specific promoter, optionally wherein the trabecular meshwork- specific promoter is a Chitinase-3-like 1 (Ch3Ll) promoter.

17. The isolated nucleic acid of claim 16, wherein the Ch3Ll promoter comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 12.

18. The isolated nucleic acid of claim 16 or 17, wherein the Ch3Ll promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12.

19. The isolated nucleic acid of any one of claims 1-18, wherein the ITRs are AAV2 ITRs.

20. A vector comprising the isolated nucleic acid of any one of claims 1-19.

21. The vector of claim 20, wherein the vector is a plasmid or a viral vector.

22. The vector of claim 21, wherein the viral vector is an adenoviral vector, an adeno- associated virus vector, a lentiviral vector, a retroviral vector, or a Baculovirus vector.

23. A composition comprising the isolated nucleic acid of any one of claims 1-19 or the vector of any one of claims 20-22.

24. The composition of claim 23, further comprising a pharmaceutically acceptable carrier.

25. A host cell comprising the isolated nucleic acid of any one of claims 1-19 or the vector of any one of claims 20-22.

26. The host cell of claim 25, wherein the host cell is a mammalian cell.

27. The host cell of claim 25 or 26, wherein the host cell is a human cell.

28. The host cell of any one of claims 25-27, wherein the host cell is a trabecular meshwork cell.

29. A recombinant adeno-associated virus (rAAV) comprising:

(i) the isolated nucleic acid of any one of claims 1-19; and

(ii) an adeno-associated virus (AAV) capsid protein.

30. The rAAV of claim 29, wherein the capsid protein has a tropism for ocular tissue.

31. The rAAV of claim 29 or 30, wherein the capsid protein is of a serotype selected from AAV2, AAV5, AAV8, AAV9, AAVrhlO, and a variant of any of the foregoing.

32. The rAAV of any one of claims 29-31, wherein the capsid protein is an AAV2 capsid protein.

33. A method for preventing or treating glaucoma in a subject, the method comprising administering to the subject the isolated nucleic acid of any one of claims 1-19, the vector of any one of claims 20-22, the composition of claim 23 or 24, or the rAAV of any one of claims 29- 32.

34. The method of claim 33, wherein the glaucoma is primary open angle glaucoma (POAG).

35. The method of claim 33 or 34, wherein the administering reduces intraocular pressure (IOP) in the subject.

36. A method of decreasing intraocular pressure (IOP) in a subject, the method comprising administering to the subject the isolated nucleic acid of any one of claims 1-19, the vector of any one of claims 20-22, the composition of claim 23 or 24, or the rAAV of any one of claims 29- 32.

37. The method of any one of claims 33-36, wherein the administering is done via ocular injection.

38. The method of claim 37, wherein the ocular injection is an intracameral injection.

39. The method of any one of claims 33-38, wherein the administering decreases RhoA expression in ocular tissue of the subject.

40. The method of any one of claims 33-39, wherein the administering decreases Rho- associated serine/threonine kinase (ROCK) signaling in ocular tissue of the subject.

41. The method of any one of claims 33-40, wherein the administering decreases accumulation of aqueous humor (AH) in an eye of the subject.

42. The method of any one of claims 33-41, wherein the administering decreases contractility of a trabecular meshwork cell in an eye of the subject.

43. A method of treating glaucoma in a subject, the method comprising administering to the subject an isolated nucleic acid comprising a transgene encoding an inhibitory nucleic acid targeting a RhoA messenger RNA (mRNA), wherein the transgene is flanked by adeno- associated virus (AAV) inverted terminal repeats (ITRs).

44. The method of claim 43, wherein the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26.

45. A method of treating glaucoma in a subject, the method comprising administering to the subject an isolated nucleic acid comprising an inhibitory nucleic acid having a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 16-26.

46. The method of any one of claims 43-45, wherein the inhibitory nucleic acid is an artificial microRNA (amiRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an antisense oligonucleotide (ASO).

47. The method of any one of claims 43-46, wherein the inhibitory nucleic acid is an artificial microRNA (amiRNA).

48. The method of claim 46 or 47, wherein the amiRNA comprises a miRNA backbone sequence selected from a miR-33, a miR-155, a miR-30, a miR-30A, a miR-64, or a miR-122 backbone sequence.

49. The method of any one of claims 46-48, wherein the amiRNA comprises a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-11.

50. The method of any one of claims 43-49, wherein the inhibitory nucleic acid comprises a nucleic acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 19, 24, or 25.

51. The method of any one of claims 43-50, wherein the inhibitory nucleic acid is an amiRNA comprising a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 9, or 10.

52. The method of any one of claims 43-51, wherein the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50.

53. The method of any one of claims 43-52, wherein the RhoA mRNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

54. The method of any one of claims 43-53, wherein the inhibitory nucleic acid is complementary to a segment of a nucleic acid sequence set forth in any one of SEQ ID NOs: 14, 15, 49, or 50.

55. The method of claim 54, wherein the segment of nucleic acid comprises 2-24 continuous nucleotides.

56. The method of any one of claims 43-55, wherein the inhibitory nucleic acid is complementary to a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-48.

57. The method of any one of claims 43-56, wherein the nucleic acid encoding the inhibitory nucleic acid is operably linked to a promoter.

58. The method of claim 57, wherein the promoter is a trabecular meshwork- specific promoter, optionally wherein the trabecular meshwork-specific promoter is a Chitinase- 3 -like 1 (Ch3Ll) promoter.

59. The method of claim 58, wherein the Ch3Ll promoter comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 12.

60. The method of claim 58 or 59, wherein the Ch3Ll promoter comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12.

61. The method of any one of claims 43-60, wherein the ITRs are AAV2 ITRs.

62. The method of any one of claims 43-61, wherein the glaucoma is primary open angle glaucoma (POAG).

63. The method of any one of claims 43-62, wherein the administering is done via ocular injection.

64. The method of claim 63, wherein the ocular injection is an intracameral injection.

65. The method of any one of claims 43-64, wherein the administering reduces intraocular pressure (IOP) in the subject.

66. The method of any one of claims 43-65, wherein the administering decreases RhoA expression in ocular tissue of the subject.

67. The method of any one of claims 43-66, wherein the administering decreases Rho- associated serine/threonine kinase (ROCK) signaling in ocular tissue of the subject.

68. The method of any one of claims 43-67, wherein the administering decreases accumulation of aqueous humor (AH) in an eye of the subject.

69. The method of any one of claims 43-68, wherein the administering decreases contractility of a trabecular meshwork cell in an eye of the subject.