Compositions For and Methods of Improving Gene Therapy

II. REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing submitted 25 Feb. 2022 as a text file named “21_2023_WO_Sequence_Listing”, created on 25 Feb. 2022 and having a size of 208 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

III. BACKGROUND

Gene transfer using recombinant adeno-associated virus (rAAV) vectors has been successfully used to treat hemophilia, inherited vision loss, and spinal muscular atrophy. The development of new rAAV vectors for the treatment of a wide range of genetic diseases, such as neurological disorders and muscular dystrophies, is advancing at an unprecedented rate. However, suboptimal transgene expression using recombinant AAV vectors is a significant challenge facing the field of gene therapy. This has necessitated the use of very high viral doses in clinical trials, leading to toxic side effects and even some patient deaths. Thus, new strategies to increase transgene expression are needed to enhance AAV gene therapy efficacy and allow for lower, safer dosing regimens.

Currently, there are no disease-modifying therapies for the many, many genetic diseases and disorders that plague the human population. Thus, there remains an urgent need for a minimally invasive, definitive therapy to address the underlying cause of as well as the sequelae of symptoms associated with these various genetic diseases and disorders. Consequently, the present disclosure provides compositions for and methods of improving mRNA transcript stability, improving mRNA transcript translation efficiency, enhancing therapy, and treating and/or preventing a disease and/or disorder, which can be used alone or in combination with other treatments.

IV. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the secondary structure of the DENV2 sfRNA including 2 xrRNAs, 2 DBs, and a 3′ SL. Various structural elements are noted. CS1/3′CYC indicates conserved sequence 1 with the 3′ cyclization sequence. CS2 indicates conserved sequence 2 while CS3 indicates conserved sequence. FIG. 1B shows the sequence conservation of the xrRNA element of subgenomic Flaviviral RNAs for DENV 1 and DENV 2, JEV 1 and JEV 2, MVEV 1 and MVEV 2, WNV2 1 and WNV 2, YFV, and ZIKV 1 and ZIKV 2. FIG. 1C shows the sequence conservation of the DB element of subgenomic Flaviviral RNAs for DENV, JEV, MVEV, WNV2, YFV, and ZKV. FIG. 1D show the sequence conservation of the 3′ SL of subgenomic Flaviviral RNAs for DENV2, JEV, MVEV, WNV2, YFV, and ZIKV.

FIG. 2A-FIG. 2B show that XRN1 restricts AAV transduction. FIG. 2A shows Huh7 cells following infection with lentivirus packaging Cas9 and either a Scramble gRNA (top) or gRNA against the XRN1 gene (bottom). Following antibiotic selection, sequencing at the XRN1 locus confirmed Cas9 activity with the XRN1-1 gRNA. The arrow denotes the predicted Cas9 cleavage site. FIG. 2B shows western blotting performed on two independent Scramble and XRN1 KO clonal cell lines for XRN1 (top) and R-actin (bottom). FIG. 2C shows the quantitation of transgene expression from different AAV serotypes in XRN1 KO cells.

FIG. 3A-FIG. 3E show that subgenomic Flaviviral RNAs increased AAV transduction efficiency. FIG. 3A shows a diagram of an experimental construct. Here, a luciferase transgene with SV40 poly-A signal and driven by the CBA promoter was placed between AAV2 inverted terminal repeats (ITRs). SfRNA (or WPRE) sequences were placed as the 3′ UTR of the luciferase mRNA. FIG. 3B shows luciferase expression in Huh7 cells transduced with the indicated constructs at 10,000 vg/cell and harvested at 3 days post-transduction. Lysates were measured for Relative Luciferase Expression (RLUs). FIG. 3C shows RNA from replicate samples was used to perform qRT-PCR for luciferase mRNAs. FIG. 3D shows mRNA expression in various cell lines were transduced with 10,000 vg/cell of the DENV2 sfRNA construct and harvested at 3 days post-transduction. Lysates were measured for Relative Luciferase Expression (RLUs). FIG. 3E shows RNA from replicate samples was used to perform qRT-PCR for luciferase mRNAs. For all graphs, samples are plotted relative to a CBA-Luc transgene with no additional 3′ UTR. Student's t-test was performed to test for statistical significance. Where indicated for FIG. 3B-FIG. 3E, * =p<0.05; ** =p<0.005; *** =p<0.0005; **** =p<0.00005.

FIG. 4A-FIG. 4C shows that the effect of DENV2 sfRNA on AAV transduction was context-dependent. FIG. 4A shows a construct in which the DENV2 sfRNA was placed in the 5′ of the transgene (top construct), 3′ of the transgene (middle construct), or as a separate and U6-driven RNA packaged in the same AAV genome (lower construct). FIG. 4B shows northern blots of HEK293 cells transfected with the various constructs in FIG. 4A and then harvested 3 days post-transfection. The northern blots were probed for either luciferase or sfRNA sequences. FIG. 4C-FIG. 4D show Huh7 cells transduced with the indicated constructs at 10,000 vg/cell and then harvested 3 days post-transfection. FIG. 4C shows lysates that were measured for Relative Luciferase Expression (RLUs) while FIG. 4D shows RNA from replicate samples that was used to perform qRT-PCR for luciferase mRNAs. Data was relative to a CBA-Luc transgene with no additional 3′ UTR. For FIG. 4C-FIG. 4D, Student's t-test was performed to test for statistical significance, and where indicated, * =p<0.05; ** =p<0.005; *** =p<0.0005; **** =p<0.00005.

FIG. 5A-FIG. 5E shows that the presence of polyA tail was necessary for increased transgene expression. FIG. 5A shows a diagram of luciferase mRNAs with either a poly-A or RiboJ (hammerhead ribozyme) 3′ end with and without the DENV2 sfRNA. FIG. 5B shows northern blots of HEK293 cells following transfection with the indicated constructs and then harvested 3 days post-transfection. The northern blots were probed for luciferase sequences. FIG. 5C shows Huh7 cells transduced with the indicated constructs at 10,000 vg/cell and then harvested at 3 days post-transduction. Lysates were measured for Relative Luciferase Expression (RLUs). FIG. 5D shows qRT-PCR for luciferase mRNAs using replicate samples. Data is shown relative to a CBA-Luc transgene with no additional 3′ UTR. FIG. 5E shows the ratio of protein and RNA expression calculated for the DENV2 RiboJ construct relative to CBA-Luc with a poly-A tail. Student's t-test was performed to test for statistical significance. In FIG. 5C-FIG. 5E, where indicated, * =p<0.05; ** =p<0.005; *** =p<0.0005; **** =p<0.00005.

FIG. 6A-FIG. 6F shows that the DENV2 DB elements were necessary and sufficient to increase AAV transduction efficiency. FIG. 6A shows a diagram of the DENV2 sfRNA with the indicated deletions of RNA structural elements. FIG. 6B shows Huh7 cells transduced with the indicated constructs at 10,000 vg/cell and harvested at 3 days post-transduction. Lysates were measured for Relative Luciferase Expression (RLUs). FIG. 6C shows qRT-PCR for luciferase mRNAs performed on replicate samples. Data is shown relative to a CBA-Luc transgene with no additional 3′ UTR. FIG. 6D shows mutations in the first DB of DENV2 that were created at the indicated locations. Huh7 cells were transduced with the indicated constructs at 10,000 vg/cell and harvested at 3 days post-transduction. FIG. 6E shows Relative Luciferase Expression (RLUs) from lysates and FIG. 6F shows qRT-PCR for luciferase mRNAs performed on replicate samples. Data is shown relative to a CBA-Luc transgene with no additional 3′ UTR. Student's t-test was performed to test for statistical significance. In FIG. 6B-FIG. 6C and FIG. 6E-FIG. 6F, where indicated, * =p<0.05; ** =p<0.005; *** =p<0.0005; **** =p<0.00005.

FIG. 7A-FIG. 7D show that DENV2 sfRNA and DB elements increase mRNA half-life. HEK293 cells were transduced with the indicated constructs at 10,000 vg/cell. At 3 days post-transduction, Actinomycin D was added to cells and RNA collected at the indicated time points. RNA levels were measured by qRT-PCR, and graphed relative to the level at 0 hours and normalized to GAPDH mRNA levels. FIG. 7A shows the fraction RNA remaining for 3 constructs. FIG. 7B shows that the DENV2 DB increased the mRNA half-life while FIG. 7C shows that the DENV2 sfRNA also increased the mRNA half-life. FIG. 7D shows that DENV2 sfRNA and DENV2 DB generated RNA having longer half-lives. Student's t-test was performed to test for statistical significance. In FIG. 7A-FIG. 7D, where indicated, * =p<0.05; ** =p<0.005; *** =p<0.0005; **** =p<0.00005.

V. BRIEF SUMMARY

Disclosed herein is an isolated nucleic acid molecule, comprising: one or more Flavivirus genetic elements. Disclosed herein is an isolated nucleic acid molecule, comprising one or more subgenomic Flavivirus RNA (sfRNA) elements.

Disclosed herein is an isolated nucleic acid molecule, comprising one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein the one or more sfRNA elements comprise a Flavivirus XRN1-resistant RNA (xrRNA) element, a Flavivirus sfRNA dumbbell (DB) RNA element, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is an isolated nucleic acid molecule, comprising a Flavivirus 3′ untranslated region (3′ UTR) element, wherein the 3′ UTR comprises one or more Flavivirus XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is an isolated nucleic acid molecule, comprising one or more Flavivirus XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, one or more Flavivirus 3′ stem loop (3′ SL) elements, or any combination.

Disclosed herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more Flavivirus genetic elements. Disclosed herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more subgenomic Flavivirus RNA (sfRNA) elements.

Disclosed herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein the one or more sfRNA elements comprise a Flavivirus XRN1-resistant RNA (xrRNA) element, a Flavivirus sfRNA dumbbell (DB) RNA element, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is an expression cassette, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule operably linked to a promoter, and a nucleic acid sequence encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein the one or more sfRNA elements comprise a Flavivirus XRN1-resistant RNA (xrRNA) element, a Flavivirus sfRNA dumbbell (DB) RNA element, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is a vector, comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a vector, comprising an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more Flavivirus genetic elements. Disclosed herein is a vector, comprising an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more subgenomic Flavivirus RNA (sfRNA) elements.

Disclosed herein is a vector, comprising an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein the 3′ UTR comprises one or more Flavivirus XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is a vector, comprising an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more Flavivirus XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, one or more Flavivirus 3′ stem loop (3′ SL) elements, or any combination.

Disclosed herein is a pharmaceutical formulation comprising an isolated nucleic acid molecule, comprising a Flavivirus 3′ untranslated region (3′ UTR) element, wherein the 3′ UTR comprises one or more Flavivirus XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is a pharmaceutical formulation comprising a disclosed vector comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein the one or more sfRNA elements comprise a Flavivirus XRN1-resistant RNA (xrRNA) element, a Flavivirus sfRNA dumbbell (DB) RNA element, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is a pharmaceutical formulation comprising a disclosed vector comprising an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide or an RNA molecule, and a nucleic acid sequence encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein the 3′ UTR comprises one or more Flavivirus XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

Disclosed herein is a method of improving mRNA transcript stability, comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein expression of the nucleic acid molecule improves mRNA transcript stability.

Disclosed herein is a method of improving mRNA transcript stability, comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, wherein expression of the nucleic acid molecule improves mRNA transcript stability.

Disclosed herein is a method of improving mRNA transcript stability, comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein expression of the nucleic acid molecule improves mRNA transcript stability.

Disclosed herein is a method of improving mRNA transcript stability, comprising administering to a subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein expression of the nucleic acid molecule improves mRNA transcript stability.

Disclosed herein is a method of improving mRNA transcript stability, comprising administering to a subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, wherein expression of the nucleic acid molecule improves mRNA transcript stability.

Disclosed herein is a method of improving mRNA transcript stability, comprising administering to a subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein expression of the nucleic acid molecule improves mRNA transcript stability.

Disclosed herein is a method of improving the mRNA transcript translation efficiency, comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein expression of the nucleic acid molecule improves mRNA transcript translation efficiency.

Disclosed herein is a method of improving the mRNA transcript translation efficiency, comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, wherein expression of the nucleic acid molecule improves mRNA transcript translation efficiency.

Disclosed herein is a method of improving the mRNA transcript translation efficiency, comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein expression of the nucleic acid molecule improves mRNA transcript translation efficiency.

Disclosed herein is a method of improving the mRNA transcript translation efficiency, comprising administering to a subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein expression of the nucleic acid molecule improves mRNA transcript translation efficiency.

Disclosed herein is a method of improving the mRNA transcript translation efficiency, comprising administering to a subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, wherein expression of the nucleic acid molecule improves mRNA transcript translation efficiency.

Disclosed herein is a method of improving the mRNA transcript translation efficiency, comprising administering to a subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein expression of the nucleic acid molecule improves mRNA transcript translation efficiency.

Disclosed herein is a method of enhancing gene therapy, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein expression of the nucleic acid molecule improves mRNA transcript stability and/or improves mRNA transcript translation efficiency.

Disclosed herein is a method of enhancing gene therapy, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, wherein expression of the nucleic acid molecule improves mRNA transcript stability and/or improves mRNA transcript translation efficiency.

Disclosed herein is a method of enhancing gene therapy, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein expression of the nucleic acid molecule improves mRNA transcript stability and/or improves mRNA transcript translation efficiency.

Disclosed herein is a method of enhancing gene therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, wherein expression of the nucleic acid molecule improves transgene mRNA stability and/or improves mRNA transcript translation efficiency.

Disclosed herein is a method of enhancing gene therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, wherein expression of the nucleic acid molecule improves transgene mRNA stability and/or improves mRNA transcript translation efficiency.

Disclosed herein is a method of enhancing gene therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, wherein expression of the nucleic acid molecule improves transgene mRNA stability and/or improves mRNA transcript translation efficiency.

Disclosed herein is a method of treating and/or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, and enhancing gene therapy through improved mRNA transcript mRNA stability and/or improved mRNA transcript translation efficiency.

Disclosed herein is a method of treating and/or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, and enhancing gene therapy through improved mRNA transcript mRNA stability and/or improved mRNA transcript translation efficiency.

Disclosed herein is a method of treating and/or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, and enhancing gene therapy through improved transgene mRNA stability and/or improved mRNA transcript translation efficiency.

Disclosed herein is a method of treating and/or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding a Flavivirus 3′ untranslated region (3′ UTR) element, and enhancing gene therapy through improved transgene mRNA stability and/or improved mRNA transcript translation efficiency.

Disclosed herein is a method of treating and/or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount a vector comprising of an isolated nucleic acid molecule encoding a transgene and encoding one or more Flavivirus genetic elements, and enhancing gene therapy through improved transgene mRNA stability and/or improved mRNA transcript translation efficiency.

Disclosed herein is a method of treating and/or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule encoding a transgene and encoding one or more subgenomic Flavivirus RNA (sfRNA) elements, and enhancing gene therapy through improved transgene mRNA stability and/or improved mRNA transcript translation efficiency.

VI. DETAILED DESCRIPTION

The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. Definitions

Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

The phrase “consisting essentially of” limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method.

The phrase “consisting of” excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ±10% of the stated value.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

As used herein, “derived from” can mean “derived from”, “based on”, “obtained from”, “obtained from” or “isolated from” depending on the context. For example, in an aspect, the term “derived from” can mean that an amino acid sequence is derived from the parent amino acid sequence by introducing a modification to at least one position. Thus, the derived amino acid sequence differs from the corresponding parent amino acid sequence in at least one corresponding position (numbering based on Kabat's EU index numbering system for the antibody Fc region). In an aspect, a disclosed amino acid sequence derived from the parent amino acid sequence can differ by 1 to 15 amino acid residues at corresponding positions. In an aspect, a derived amino acid sequence can have a high degree sequence identity to its parent amino acid sequence. In an aspect, a disclosed amino acid sequence derived from the parent amino acid sequence can have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more sequence identity to the parent or original sequence.

As used herein, the term “subject” refers to the target of administration, e.g., a human being. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease or disorder, be suspected of having a disease or disorder, or be at risk of developing a disease or disorder (e.g., a genetic disease or disorder).

As used herein, the term “diagnosed” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “diagnosed with a disease or disorder” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can be treated by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “suspected of having a disease or disorder” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can likely be treated by one or more of by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.

A “patient” refers to a subject afflicted with a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder.

As used herein, the phrase “identified to be in need of treatment for a disease or disorder,” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., a genetic disease or disorder) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the genetic disease or disorder. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

As used herein, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having a disease or disorder such as a genetic disease or disorder). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level.

The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a genetic disease or disorder) a or related complication from progressing to that complication.

As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, and/or a disclosed RNA therapeutic can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more route.

In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to treat or prevent a disease or disorder (such as genetic disease or disorder). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof.

By “determining the amount” is meant both an absolute quantification of a particular analyte (e.g., an mRNA sequence containing a particular tag) or a determination of the relative abundance of a particular analyte (e.g., an amount as compared to a mRNA sequence including a different tag). The phrase includes both direct or indirect measurements of abundance (e.g., individual mRNA transcripts may be quantified or the amount of amplification of an mRNA sequence under certain conditions for a certain period may be used a surrogate for individual transcript quantification) or both.

As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, by changing the duration of time one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject, or by substituting for one or more of the disclosed components and/or reagents with a similar or equivalent component and/or reagent. The same applies to all disclosed therapeutic agents, immune modulators, immunosuppressive agents, proteosome inhibitors, etc.

As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

The term “contacting” as used herein refers to bringing one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof together with a target area or intended target area in such a manner that the one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof exert an effect on the intended target or targeted area either directly or indirectly. A target area or intended target area can be one or more of a subject's organs (e.g., lungs, heart, liver, kidney, brain, etc.). In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as a genetic disease or disorder). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a genetic disease or disorder).

As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a genetic disease or disorder. Methods and techniques used to determine the presence and/or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, a genetic disease or disorder).

As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a disease or disorder). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a genetic disease or disorder). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and/or mutant protein or enzyme.

As used herein, “RNA therapeutics” can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2′-O-Me-RNA, 2′-MEO-RNA, 2′-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (lncRNA), such as, for example, a long intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

As used herein, “lipid nanoparticles” or “LNPs” can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be use interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.

As used herein, “small molecule” can refer to any organic or inorganic material that is not a polymer. Small molecules exclude large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). In an aspect, a “small molecule”, for example, can be a drug that can enter cells easily because it has a low molecular weight. In an aspect, a small molecule can be used in conjunction with a disclosed composition in a disclosed method.

As used herein, “promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

“Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters.

“Liver-specific promoters” are known to the art and include, but are not limited to, the thyroxin binding globulin (TBG) promoter, the α1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the α-1-anti-trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human α1-antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC172 promoter comprising the hAAT promoter and the α1-microglobulin enhancer, the DC190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In an aspect, a liver specific promoter can comprise about 845-bp and comprise the thyroid hormone-binding globulin promoter sequences (2382 to 13), two copies of enhancer sequences (22,804 through 22,704), and a 71-bp leader sequence as described by Ill C R, et al. (1997). In an aspect, a disclosed liver specific promoter can comprise the sequence set forth in SEQ ID NO:32, or a sequence having about 50%, about 60%, about 70% about 80%, about 90%, about 95%, or more identity to the sequence set forth in SEQ ID NO:32.

Ubiquitous/constitutive promoters” are known to the art and include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-α (EF1-α) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PγK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, β-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous/constitutive promoters.

As used herein, an “inducible promoter” refers to a promoter that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness can be determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).

As used herein, “tropism” refers to the specificity of an AAV capsid protein present in an AAV viral particle, for infecting a particular type of cell or tissue. The tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles comprising the hybrid AAV capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well-known in the art such as those disclosed in the examples of the present application. As used herein, the term “liver tropism” or “hepatic tropism” refers to the tropism for liver or hepatic tissue and cells, including hepatocytes.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and/or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3′- and/or 5′-side are 100% identical.

As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes.urv.es/OPTIMIZER/).

As used herein, “CRISPR or clustered regularly interspaced short palindromic repeat” is an ideal tool for correction of genetic abnormalities as the system can be designed to target genomic DNA directly. A CRISPR system involves two main components—a Cas9 enzyme and a guide (gRNA). The gRNA contains a targeting sequence for DNA binding and a scaffold sequence for Cas9 binding. Cas9 nuclease is often used to “knockout” target genes hence it can be applied for deletion or suppression of oncogenes that are essential for cancer initiation or progression. Similar to ASOs and siRNAs, CRISPR offers a great flexibility in targeting any gene of interest hence, potential CRISPR based therapies can be designed based on the genetic mutation in individual patients. An advantage of CRISPR is its ability to completely ablate the expression of disease genes which can only be suppressed partially by RNA interference methods with ASOs or siRNAs. Furthermore, multiple gRNAs can be employed to suppress or activate multiple genes simultaneously, hence increasing the treatment efficacy and reducing resistance potentially caused by new mutations in the target genes.

As used herein, “CRISPR-based endonucleases” include RNA-guided endonucleases that comprise at least one nuclease domain and at least one domain that interacts with a guide RNA. As known to the art, a guide RNA directs the CRISPR-based endonucleases to a targeted site in a nucleic acid at which site the CRISPR-based endonucleases cleaves at least one strand of the targeted nucleic acid sequence. As the guide RNA provides the specificity for the targeted cleavage, the CRISPR-based endonuclease is universal and can be used with different guide RNAs to cleave different target nucleic acid sequences. CRISPR-based endonucleases are RNA-guided endonucleases derived from CRISPR/Cas systems. Bacteria and archaea have evolved an RNA-based adaptive immune system that uses CRISPR (clustered regularly interspersed short palindromic repeat) and Cas (CRISPR-associated) proteins to detect and destroy invading viruses or plasmids. CRISPR/Cas endonucleases can be programmed to introduce targeted site-specific double-strand breaks by providing target-specific synthetic guide RNAs (Jinek et al. (2012) Science. 337:816-821).

In an aspect, a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, or type III system. Non-limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.

In an aspect, a disclosed CRISPR-based endonuclease can be derived from a type II CRISPR/Cas system. For example, in an aspect, a CRISPR-based endonuclease can be derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. In an aspect, the CRISPR-based nuclease can be derived from a Cas9 protein from Staphylococcus Aureus (SEQ ID NO:33 (nucleic acid) or SEQ ID NO:34 (amino acid) or Streptococcus pyogenes (SEQ ID NO:35).

In general, CRISPR/Cas proteins can comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains can interact with the guide RNA such that the CRISPR/Cas protein is directed to a specific genomic or genomic sequence. CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, as well as other domains.

The CRISPR-based endonuclease can be a wild type CRISPR/Cas protein (such as for example, Staphylococcus Aureus (SEQ ID NO:33 (nucleic acid) or SEQ ID NO:34 (amino acid) or Streptococcus pyogenes (SEQ ID NO:35), a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein. The CRISPR/Cas protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein. For example, in an aspect, nuclease (i.e., DNase, RNase) domains of the CRISPR/Cas protein can be modified, deleted, or inactivated. A CRISPR/Cas protein can be truncated to remove domains that are not essential for the function of the protein. A CRISPR/Cas protein also can be truncated or modified to optimize the activity of the protein or an effector domain fused with a CRISPR/Cas protein.

In an aspect, a disclosed CRISPR-based endonuclease can be derived from a wild type Cas9 protein (such as, for example, SEQ ID NO:33) or fragment thereof. In an aspect, a disclosed CRISPR-based endonuclease can be derived from a modified Cas9 protein. For example, the amino acid sequence of a disclosed Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.

As used herein, “immune tolerance,” “immunological tolerance,” and “immunotolerance” refers to a state of unresponsiveness or blunted response of the immune system to substances (e.g., a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed transgene product, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, etc.) that have the capacity to elicit an immune response in a subject. Immune tolerance is induced by prior exposure to a specific antigen. Immune tolerance can be determined in a subject by measuring antibodies against a particular antigen or by liver-restricted transgene expression with a viral vector (such as, for example, AAV). Low or absent antibody titers over time is an indicator of immune tolerance. For example, in some embodiments, immune tolerance can be established by having IgG antibody titers of less than or equal to about 12,000, 11,500, 11,000, 10,500, 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, or 6,000 within following gene therapy (such as the administration of the transgene encoding, for example, a missing, deficient, and/or mutant protein or enzyme).

As known to the art, antibodies (Abs) can mitigate AAV infection through multiple mechanisms by binding to AAV capsids and blocking critical steps in transduction such as cell surface attachment and uptake, endosomal escape, productive trafficking to the nucleus, or uncoating as well as promoting AAV opsonization by phagocytic cells, thereby mediating their rapid clearance from the circulation. For example, in humans, serological studies reveal a high prevalence of NAbs in the worldwide population, with about 67% of people having antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5 through 9. Vector immunogenicity represents a major challenge in re-administration of AAV vectors.

In an aspect, also disclosed herein are partial self-complementary parvovirus (e.g., a disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., a disclosed AAV) particles including such genomes. In an aspect, provided herein is a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome such as for example, a disclosed AAV. In an aspect, provided herein is a partial self-complementary parvovirus genome including a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. A self-complementary region can comprise a nucleotide sequence that is complementary to the payload construct. A disclosed self-complementary region can have a length that is less the entire length of the payload construct.

In an aspect, a disclosed self-complementary region of a disclosed parvovirus genome can comprise a minimum length, while still having a length that is less the entire length of the payload construct. In an aspect, a disclosed self-complementary region can comprise at least 50 bases in length, at least 100 bases in length, at least 200 in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, at least 900 bases in length, or at least 1,000 bases in length.

In an aspect, a “self-complementary parvovirus genome” can be a single stranded polynucleotide having, in the 5′ to 3′ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct comprising, for example, a desired gene), a second parvovirus ITR sequence, a second heterologous sequence, wherein the second heterologous sequence is complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a “partial self-complementary genome” does not include three parvovirus ITRs and the second heterologous sequence that is complementary to the first heterologous sequence has a length that is less than the entire length of the first heterologous sequence (e.g., payload construct). Accordingly, a partial self-complementary genome is a single stranded polynucleotide having, in the 5′ to 3′ direction or the 3′ to 5′ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct), a second parvovirus ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.

As used herein, “immune-modulating” refers to the ability of a disclosed isolated nucleic acid molecules, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

As used herein, “immune modulator” refers to an agent that is capable of adjusting a given immune response to a desired level (e.g., as in immunopotentiation, immunosuppression, or induction of immunologic tolerance). Examples of immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-Rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGF1R antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, orally, transcutaneously, intraperitoneally (IP), or intravaginally. In an aspect, a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes.

As used herein, the term “immunotolerant” refers to unresponsiveness to an antigen (e.g., a vector, a therapeutic protein, a transgene product, etc.). An immunotolerant promoter can reduce, ameliorate, or prevent transgene-induced immune responses that can be associated with gene therapy. Assays known in the art to measure immune responses, such as immunohistochemical detection of cytotoxic T cell responses, can be used to determine whether one or more promoters can confer immunotolerant properties.

As used herein, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof) may be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy (e.g., agent) to a subject having or diagnosed with a disease or disorder (such as a genetic disease or disorder).

Disclosed are the components to be used to prepare the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

B. Compositions for Use in the Disclosed Methods
1. Nucleic Acid Molecules

Disclosed herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a transgene; and a nucleic acid sequence encoding one or more Flavivirus genetic elements, wherein the Flavivirus genetic elements comprise one or more Flavivirus 3′ untranslated regions (3′ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, one or more Flavivirus 3′ stem loop (3′ SL) elements, or any combination thereof.

Disclosed herein is an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding one or more Flavivirus genetic elements, wherein the Flavivirus genetic elements comprise one or more Flavivirus 3′ untranslated regions (3′ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more XRN1-resistant RNA (xrRNA) elements, one or more Flavivirus sfRNA dumbbell (DB) RNA elements, one or more Flavivirus 3′ stem loop (3′ SL) elements, or any combination thereof.

Disclosed herein is an isolated nucleic acid molecule, comprising: one or more Flavivirus genetic elements.

In an aspect, disclosed Flavivirus genetic elements can comprise a 3′ untranslated (3′ UTR) element, a subgenomic Flavivirus RNA (sfRNA) element, a Flavivirus XRN1-resistant RNA (xrRNA) element, a Flavivirus sfRNA dumbbell (DB) RNA element, a Flavivirus 3′ stem loop (3′ SL) element, or any combination thereof.

In an aspect, a disclosed Flavivirus genetic element can comprise a 3′ UTR element. For example, in an aspect, a disclosed 3′ UTR element can comprise the sequence set forth in any one of SEQ ID NO:08-SEQ ID NO:19. In an aspect, a disclosed 3′ UTR element comprises the sequence forth in SEQ ID NO:08 or a fragment thereof, SEQ ID NO:09 or a fragment thereof, SEQ ID NO:10 or a fragment thereof, SEQ ID NO:11 or a fragment thereof, SEQ ID NO:12 or a fragment thereof, SEQ ID NO:13 or a fragment thereof, SEQ ID NO:14 or a fragment thereof, SEQ ID NO:15 or a fragment thereof, SEQ ID NO:16 or a fragment thereof, SEQ ID NO:17 or a fragment thereof, SEQ ID NO:18 or a fragment thereof, SEQ ID NO:19 or a fragment thereof.

In an aspect, a disclosed 3′ UTR element can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of SEQ ID NO:08-SEQ ID NO:19. In an aspect, a disclosed 3′ UTR element can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.

In an aspect, a disclosed Flavivirus genetic element can comprise a 3′ UTR element. For example, in an aspect, a disclosed 3′ UTR element can comprise the sequence set forth in any one of GenBank Accession Nos. U88535.1, KU725663.1, JQ411814.1, KJ160504.1, M55506.1, NC000943.1, KU886216.1, NC007580.2, U27495.1, M12294.2, KJ776791.2, MT107250.1. In an aspect, a disclosed 3′ UTR element can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of GenBank Accession Nos. U88535.1, KU725663.1, JQ411814.1, KJ160504.1, M55506.1, NC000943.1, KU886216.1, NC007580.2, U27495.1, M12294.2, KJ776791.2, MT107250.1. In an aspect, a disclosed 3′ UTR element can comprise a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100^%identity to the sequence set forth in GenBank Accession Nos. U88535.1, KU725663.1, JQ411814.1, KJ160504.1, M55506.1, NC000943.1, KU886216.1, NC007580.2, U27495.1, M12294.2, KJ776791.2, MT107250.1.

In an aspect, a disclosed 3′ UTR element can comprise a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100^%identity to the sequence set forth in any one of SEQ ID NO:08-SEQ ID NO: 19. In an aspect, a disclosed 3′ UTR element can comprise a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in SEQ ID NO:09, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.