Methods and compositions for increasing protein expression and / or treating haploinsufficiency disorder

Abstract

To provide methods to increase expression in a mammalian cell of a gene product encoded by a gene containing a non-optimal codon.SOLUTION: A method comprises introducing into a mammalian cell an effective amount of a first exogenous tRNA that (i) comprises an anticodon that hybridizes to a first non-optimal codon and (ii) is capable of being aminoacylated with an amino acid, wherein introduction of the first exogenous tRNA into the cell increases the expression of the gene product relative to a similar cell lacking the first exogenous tRNA.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 736,847, filed September 26, 2018, the subject matter of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to methods and compositions for increasing expression of a gene product encoded by a gene that contains non-optimal codons and / or for treating a haploinsufficiency disorder in a subject. [Background technology]

[0003] Haploinsufficiency occurs when one allele of a gene is inactivated and the amount of gene product expressed from the remaining active allele is insufficient for proper gene function. Many disorders are associated with or caused by haploinsufficiency.

[0004] An example of a haploinsufficiency disorder is Dravet syndrome. Dravet syndrome is a rare and devastating form of intractable epilepsy that begins in infancy. Initially, patients experience prolonged seizures. In the second year, additional types of seizures begin to occur, which usually coincide with a decline in development, probably due to repeated cerebral hypoxia. This leads to poor development of language and motor skills. Mutations in the SCN1A (encoding voltage-gated sodium channel α subunit), SCN1B (encoding voltage-gated sodium channel β1 subunit), SCN2A, SCN3A, SCN9A, GABRG2 (encoding gamma-aminobutyric acid receptor γ2 subunit), GABRD (encoding gamma-aminobutyric acid receptor Δ subunit) and / or PCDH19 genes have been associated with Dravet syndrome.

[0005] For example, Dravet syndrome is caused by mutations in the SCN1A gene, in which one SCN1A allele is inactivated, resulting in insufficient or reduced amounts of functional SCN1A gene product. The SCN1A gene normally encodes the neuronal voltage-gated sodium channel α subunit, Na(V)1.1. In mouse models, loss-of-function mutations in SCN1A have been observed to result in reduced sodium currents and impair the excitability of hippocampal GABAergic interneurons.

[0006] Thus, there is a need in the art for improved compositions and methods for increasing protein expression and treating diseases mediated by haploinsufficiency, including Dravet Syndrome. Summary of the Invention

[0007] The present disclosure generally relates to methods and compositions for increasing expression of gene products encoded by genes containing non-optimal codons and / or for treating haploinsufficiency disorders in subjects. The presence of non-optimal codons in a gene of interest can result in destabilization of mRNA transcribed from the gene, affecting the production of functional proteins encoded by the gene. In such a situation, destabilization of mRNA transcripts can occur, for example, when the abundance of a particular aminoacylated tRNA molecule hybridizing to the non-optimal codon is less than other different tRNAs available in the cell that are aminoacylated with the same amino acid. Surprisingly, it has been discovered that increasing the abundance of such tRNAs in a cell compared to other naturally abundant tRNAs can promote the expression and / or amount of functional proteins produced by the cell. Expression of a gene product, such as a protein, can be increased and / or selectively increased (i.e., expression of a gene product can be increased over another gene product or over any other gene product) by introducing into a cell a minimum number (e.g., 1, 2, or 3) of tRNAs that decode the non-optimal codons in the gene.

[0008] Similarly, for a subject having a gene that includes a non-optimal codon, administering to the subject an effective amount of tRNA that decodes the non-optimal codon can increase the expression of gene products from the gene in the subject.Accordingly, administering to the subject tRNA can treat diseases mediated by insufficient expression of gene products in the subject, such as haploinsufficiency disorders.Furthermore, the features of the methods and compositions described herein make them desirable for use in treating disorders in human subjects, such as haploinsufficiency disorders.For example, tRNA is relatively small in size and can be produced by recombinant technology, and the methods and compositions described herein can treat haploinsufficiency disorders regardless of the mutation or mechanism by which the allele is inactivated.

[0009] Thus, in one embodiment, the disclosure provides a method for increasing expression in a mammalian cell of a gene product encoded by a gene that includes a first non-optimal codon, the method comprising: (i) introducing into the cell an effective amount of a first exogenous tRNA that includes an anticodon that hybridizes to the first non-optimal codon and (ii) is capable of being aminoacylated with an amino acid, wherein introducing the first exogenous tRNA into the cell increases expression of the gene product compared to a similar cell lacking the first exogenous tRNA.

[0010] In certain embodiments, the gene includes a second non-optimal codon and the method further comprises introducing into the cell an effective amount of a second exogenous tRNA that (i) includes an anticodon that hybridizes to the second non-optimal codon and ii) is capable of being aminoacylated with an amino acid, whereby introduction of the second exogenous tRNA into the cell increases expression of the gene product compared to a similar cell lacking the second exogenous tRNA. In certain embodiments, the gene includes a third non-optimal codon and the method further comprises introducing into the cell an effective amount of a third exogenous tRNA that (i) includes an anticodon that hybridizes to the third non-optimal codon and ii) is capable of being aminoacylated with an amino acid, whereby introduction of the third exogenous tRNA into the cell increases expression of the gene product compared to a similar cell lacking the third exogenous tRNA.

[0011] In another embodiment, the disclosure provides a method for increasing expression in a mammalian cell of a gene product encoded by a gene that includes a first non-optimal codon, the method comprising: introducing into the cell an effective amount of a first expression vector capable of expressing a first exogenous tRNA, the first exogenous tRNA (i) including an anticodon that hybridizes to the first non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, wherein expression of the first exogenous tRNA in the cell increases expression of the gene product compared to a similar cell lacking the first exogenous tRNA.

[0012] In certain embodiments, the gene comprises a second non-optimal codon, and the method further comprises introducing into the cell an effective amount of a second expression vector capable of expressing a second exogenous tRNA, the second exogenous tRNA comprising (i) an anticodon that hybridizes to the second non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, wherein expression of the second exogenous tRNA in the cell increases expression of the gene product compared to a similar cell lacking the second exogenous tRNA. In certain embodiments, the gene comprises a third non-optimal codon, and the method further comprises introducing into the cell an effective amount of a third expression vector capable of expressing a third exogenous tRNA, the third exogenous tRNA comprising (i) an anticodon that hybridizes to the third non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, wherein expression of the third exogenous tRNA in the cell increases expression of the gene product compared to a similar cell lacking the third exogenous tRNA.

[0013] In certain embodiments, the first, second, and / or third expression vectors are the same. In certain embodiments, the first, second, and / or third expression vectors are viral vectors, such as adeno-associated viral (AAV) vectors.

[0014] In certain embodiments of any of the foregoing methods of increasing expression, the cell is a human cell. In certain embodiments, the cell is a central nervous system cell, e.g., a neuron.

[0015] In certain embodiments, the gene is selected from the group consisting of AGGF1, ARHGAP31, BMPR2, CHD7, COL2A1, COL3A1, CTLA4, CTNNB1, DLL4, EHMT1, ELN, ENG, FAS, FBN1, FOXG1, GATA3, GLI3, GRN, IRF6, JAG1, KCNQ4, LMX1B, MBD5, MED13L, MITF, MNX1, MYCN, NFIA, NFIX. , NOTCH1, NSD1, PAX3, PHIP, PRKAR1A, RAI1, RBPJ, RPS14, RUNX2, SALL4, SCN1A, SETBP1, SHANK3, SHH, SHOX, SLC2A1 / GLUT1, SOX10, SYNGAP1, TBX1, TBX3, TBX5, TCF4, TCOF1, TGIF1, TNXB, TRPS1, WT1, and ZIC2. In a specific embodiment, the gene is SCN1A.

[0016] In another aspect, the disclosure provides a method of treating a haploinsufficiency disorder in a subject in need thereof, the subject having a haploinsufficient gene that includes a first non-optimal codon. The method includes administering to the subject an effective amount of a first tRNA that (i) includes an anticodon that hybridizes to the first non-optimal codon, and (ii) is capable of being aminoacylated with an amino acid, thereby treating the haploinsufficiency disorder in the subject.

[0017] In certain embodiments, the haploinsufficient gene comprises a second non-optimal codon and the method further comprises administering to the subject an effective amount of a second tRNA that (i) comprises an anticodon that hybridizes to the second non-optimal codon and (ii) is capable of being aminoacylated with an amino acid. In certain embodiments, the haploinsufficient gene comprises a third non-optimal codon and the method further comprises administering to the subject an effective amount of a third tRNA that (i) comprises an anticodon that hybridizes to the third non-optimal codon and (ii) is capable of being aminoacylated with an amino acid.

[0018] In another aspect, the disclosure provides a method of treating a haploinsufficiency disorder in a subject in need thereof, the subject having a haploinsufficient gene comprising a first non-optimal codon. The method comprises administering to the subject an effective amount of a first expression vector capable of expressing a first tRNA, the first tRNA (i) comprising an anticodon that hybridizes to the first non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, thereby treating the haploinsufficiency disorder in the subject.

[0019] In certain embodiments, the haploinsufficient gene comprises a second non-optimal codon and the method further comprises administering to the subject an effective amount of a second expression vector capable of expressing a second tRNA that (i) comprises an anticodon that hybridizes to the second non-optimal codon and (ii) is capable of being aminoacylated with an amino acid. In certain embodiments, the haploinsufficient gene comprises a third non-optimal codon and the method further comprises administering to the subject an effective amount of a third expression vector capable of expressing a third tRNA that (i) comprises an anticodon that hybridizes to the third non-optimal codon and (ii) is capable of being aminoacylated with an amino acid.

[0020] In certain embodiments, the first, second, and / or third expression vectors are the same. In certain embodiments, the first, second, and / or third expression vectors are viral vectors, such as adeno-associated viral (AAV) vectors.

[0021] In certain embodiments of any of the foregoing methods of treatment, the subject is a human.

[0022] In certain embodiments, the haploinsufficiency disorder is selected from the group consisting of 5q syndrome, Adams-Oliver syndrome 1, Adams-Oliver syndrome 3, Adams-Oliver syndrome 5, Adams-Oliver syndrome 6, Alagille syndrome 1, autoimmune lymphoproliferative syndrome type IA, autoimmune lymphoproliferative syndrome type V, autosomal dominant deafness-2A, brain malformations with or without urinary tract defects (BRMUTD), Carney complex type 1, CHARGE syndrome, Cleidocranial dysplasia, Currarino syndrome, Denys-Drash syndrome / Frasier syndrome, developmental delay, intellectual disability, obesity, and dysmorphic appearance (DIDOD), DiGeorge syndrome (TBX1-associated), Dravet syndrome, Duane-radial row ray syndrome, Ehlers-Danlos syndrome (classic), Ehlers-Danlos syndrome (vascular), Feingold syndrome 1, Frontotemporal Lobar Degeneration with TDP43 Inclusions (FTLD-TDP), GRN-related, GLUT1 Deficiency Syndrome, Greig Acupuncture Polysyndactylycephalopolysyndactyly syndrome, hereditary hemorrhagic telangiectasia type 1, holoprosencephaly 3, holoprosencephaly 4, holoprosencephaly 5, Holt-Oram syndrome, hypoparathyroidism, sensorineural deafness, and kidney disease (HDR), Kleefstra syndrome 1, Klippel-Trenaunay syndrome (AAGF-associated), Leri-Weill chondrodysplasia, Marfan syndrome, mental retardation and distinctive facial features with or without heart defects (MRFACD), mental retardation, autosomal dominant 1, mental retardation, autosomal dominant 19, mental retardation, autosomal dominant 29, Nail-patella syndrome (NPS), Phelan-McDermid syndrome, Pitt-Hopkins syndrome, primary pulmonary hypertension 1, Rett syndrome (congenital mutation), Smith-Magenis syndrome (RAI1 associated), Sotos syndrome 1, Sotos syndrome 2, Stickler syndrome type I, supravalvular aortic stenosis, SYNGAP1 associated intellectual disability, Treacher Collins syndrome, trichorhinophallic syndrome type I, ulnar-mammary syndrome, van der Woude syndrome 1, Waardenburg syndrome type 1, Waardenburg syndrome type 2A, and Waardenburg syndrome type 4C. In certain embodiments, the haploinsufficiency disorder is Dravet syndrome.

[0023] In certain embodiments, the haploinsufficient genes are AGGF1, ARHGAP31, BMPR2, CHD7, COL2A1, COL3A1, CTLA4, CTNNB1, DLL4, EHMT1, ELN, ENG, FAS, FBN1, FOXG1, GATA3, GLI3, GRN, IRF6, JAG1, KCNQ4, LMX1B, MBD5, MED13L, MITF, MNX1, MYCN, NFIA, NF IX, NOTCH1, NSD1, PAX3, PHIP, PRKAR1A, RAI1, RBPJ, RPS14, RUNX2, SALL4, SCN1A, SETBP1, SHANK3, SHH, SHOX, SLC2A1 / GLUT1, SOX10, SYNGAP1, TBX1, TBX3, TBX5, TCF4, TCOF1, TGIF1, TNXB, TRPS1, WT1, and ZIC2. In a specific embodiment, the haploinsufficient gene is SCN1A.

[0024] In certain embodiments, the haploinsufficient disorder is a disorder set forth in Table 1 or Table 2 below, and the haploinsufficient gene is a gene set forth in the corresponding row of Table 1 or Table 2 below.

[0025] In certain embodiments, the method further comprises administering to the subject DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), a ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), or valproic acid.

[0026] In certain embodiments of any of the foregoing methods, the gene is a gene listed in Table 1 or Table 2 below, and the first, second, and / or third non-optimal codons are codons listed in the corresponding row of Table 1 or Table 2 below. In certain embodiments, the haploinsufficiency disorder is a disorder listed in Table 1 or Table 2 below, and the first, second, and / or third non-optimal codons are codons listed in the corresponding row of Table 1 or Table 2 below.

[0027] In certain embodiments of any of the foregoing methods, the first, second, and / or third non-optimal codon is selected from ATA, GTA, and AGA.

[0028] In certain embodiments of any of the foregoing methods, the first, second, and / or third tRNA is selected from tRNAs comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In certain embodiments, the first, second, and / or third expression vector comprises a nucleotide sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0029] In another aspect, the disclosure provides an expression vector comprising a nucleic acid encoding a first, second, and / or third tRNA that (i) comprises an anticodon that hybridizes to a third non-optimal codon, and (ii) can be aminoacylated with an amino acid. In certain embodiments, the expression vector comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3. In certain embodiments, the expression vector comprises the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and / or SEQ ID NO:11. In certain embodiments, the expression vector is a viral vector, e.g., a DNA viral vector, e.g., an adeno-associated viral (AAV) vector. In another aspect, the disclosure provides a pharmaceutical composition comprising any of the aforementioned expression vectors and a pharma- ceutically acceptable excipient.

[0030] These and other aspects and features of the present invention are set forth in the following detailed description and claims. [Brief description of the drawings]

[0031] The present invention may be more fully understood with reference to the following drawings. [Figure 1]Schematic diagram of the SCN1A mRNA transcript containing non-optimal codons that destabilize the mRNA transcript. The non-optimal codons are shown as dark regions outside the grey transcript. Expression of the indicated tRNAs stabilizes the mRNA transcript and promotes increased protein expression. [Figure 2A] Consensus tRNA secondary structure. Residue numbering is based on the tRNA numbering system described in Steinberg et al. (1993) NUCLEIC ACIDS RES. 21:3011-15. [Figure 2B] 2A is a table showing the modification profile of tRNA sequences from the cytosol of certain eukaryotic organisms. The ratios in the table indicate the frequency of occurrence of the listed nucleotides at the numbered positions shown in FIG. 2A. The abbreviations of the modified residues are defined in Motorin et al. (2005) "Transfer RNA Modification," ENCYCLOPEDIA OF LIFE SCIENCES, John Wily & Sons, Inc. [Diagram 3] Graph showing quantification of SCN1A and GAPDH mRNA levels in HEK293 cells after expression of tRNAATA, tRNAGTA, and tRNAAGA. Values ​​shown were calculated by the ΔΔCt method relative to cells expressing SCN1A but no tRNA. + indicates 100 ng DNA for each tRNA, ++ indicates 200 ng DNA for each tRNA, and +++ indicates 300 ng DNA for each tRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure generally relates to methods and compositions for increasing expression of gene products encoded by genes containing non-optimal codons and / or for treating haploinsufficiency disorders in a subject. The presence of non-optimal codons in a gene of interest can cause the mRNA transcribed from the gene to become destabilized, which can affect the production of functional proteins encoded by the gene. Under such circumstances, destabilization of mRNA transcripts can occur, for example, when the abundance of a particular aminoacylated tRNA molecule that hybridizes to the non-optimal codon is less than other different tRNAs available in the cell that are aminoacylated with the same amino acid. Without wishing to be bound by theory, it is believed that given the low abundance of certain tRNAs, those available tRNAs may take longer to reach and be incorporated into ribosomes during protein synthesis than other more abundant tRNAs, which in turn can cause a delay in the synthesis of proteins while the required amino acid is incorporated at that position. It is believed that the delay in protein synthesis rate induces a corresponding destabilization of the mRNA transcript, which can result in a decrease in the production of the protein encoded by the mRNA transcript. It has been discovered that increasing the abundance of such tRNAs in a cell of interest relative to other cells can, of course, result in the more abundant tRNAs promoting the expression and / or amount of functional protein produced by the cell.

[0033] Expression of a gene product, i.e., the product resulting from expression of a gene, e.g., an mRNA transcript encoded by a gene, a protein encoded by a gene, etc., can be increased and / or selectively increased (e.g., expression of a gene product can be increased over another gene product or over any other gene product) by introduction into a cell of a minimum number (e.g., 1, 2, or 3) of tRNAs that decode non-optimal codons in the gene.

[0034] Similarly, for a subject having a gene that includes a non-optimal codon, administering to the subject an effective amount of tRNA that decodes the non-optimal codon can increase the expression of the gene product from the gene in the subject.Accordingly, administering to the subject tRNA can treat a disease mediated by insufficient expression of a gene product in the subject, such as haploinsufficiency disorder.Furthermore, the characteristics of the methods and compositions described herein can make them desirable for use in treating disorders in human subjects, such as haploinsufficiency disorder.For example, tRNA is relatively small in size and can be produced by recombinant technology, and the methods and compositions described herein can treat haploinsufficiency disorder regardless of the mutation or mechanism by which the allele is inactivated.

[0035] Thus, in one embodiment, the disclosure provides a method for increasing expression in a mammalian cell of a gene product encoded by a gene that includes a first non-optimal codon, the method comprising: (i) introducing into the cell an effective amount of a first exogenous tRNA that includes an anticodon that hybridizes to the first non-optimal codon and (ii) is capable of being aminoacylated with an amino acid, wherein introducing the first exogenous tRNA into the cell increases expression of the gene product compared to a similar cell lacking the first exogenous tRNA.

[0036] In another embodiment, the disclosure provides a method for increasing expression in a mammalian cell of a gene product encoded by a gene that includes a first non-optimal codon, the method comprising: introducing into the cell an effective amount of a first expression vector capable of expressing a first exogenous tRNA, the first exogenous tRNA (i) including an anticodon that hybridizes to the first non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, wherein expression of the first exogenous tRNA in the cell increases expression of the gene product compared to a similar cell lacking the first exogenous tRNA.

[0037] It is further contemplated that in any of the foregoing methods, the gene may include an additional non-optimal codon (e.g., a second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth non-optimal codon), and the method may further include introducing into the cell an effective amount of an additional exogenous tRNA or expression vector (e.g., a second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth exogenous tRNA or expression vector) to increase expression of the gene product.

[0038] For example, in certain embodiments, where the gene includes a second and / or third non-optimal codon, the method further includes introducing into the cell an effective amount of a second and / or third exogenous tRNA that (i) includes an anticodon that hybridizes to the second and / or third non-optimal codon, and ii) is capable of being aminoacylated with an amino acid, wherein introduction of the second and / or third exogenous tRNA into the cell increases expression of the gene product compared to a similar cell lacking the second and / or third exogenous tRNA. In certain embodiments, where the gene comprises a second and / or a third non-optimal codon, the method further comprises introducing into the cell an effective amount of a second and / or a third expression vector capable of expressing a second and / or a third exogenous tRNA, the second and / or the third exogenous tRNA (i) comprising an anticodon that hybridizes to the second and / or the third non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, wherein expression of the second and / or the third exogenous tRNA in the cell increases expression of the gene product compared to a similar cell lacking the second and / or the third exogenous tRNA.

[0039] In certain embodiments, the methods increase expression of the gene product by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more in a cell, tissue, or subject compared to a cell, tissue, or subject lacking the exogenous tRNA. In certain embodiments, the method increases expression of a gene product in a cell, tissue, or subject by about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to 60%, 20% to 40%, 40% to 200%, 40% to 180%, 40% to 160%, 40% to 140%, 40% to 120%, 40% to 100%, 40% to 80%, 40% to 60%, 60% to 200%, 60% to 180%, 60% to 160%, 60% to 1 40%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about Increase by 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200%.In certain embodiments, the method increases expression of a gene product in a cell, tissue, or subject by about 100% to about 1000%, about 100% to about 900%, about 100% to about 800%, about 100% to about 700%, about 100% to about 600%, about 100% to about 500%, about 100% to about 400%, about 100% to about 500%, about 100% to about 600%, about 100% to about 700%, about 100% to about 800%, about 100% to about 900%, about 100% to about 10 ... 0% to about 300%, about 100% to about 200%, about 200% to about 1000%, about 200% to about 900%, about 200% to about 800%, about 200% to about 700%, about 200% to about 600%, about 200% to about 500%, about 200% to about 400%, about 200% to about 300%, about 300% to about 1000%, about 300% to about 900%, about 300% to about 800%, about 300% to 700%, 300% to 600%, 300% to 500%, 300% to 400%, 400% to 1000%, 400% to 900%, 400% to 800%, 400% to 700%, 400% to 600%, 400% to 500%, 500% to 1000%, 500% to 900%, 500% to 800% , about 500% to about 700%, about 500% to about 600%, about 600% to about 1000%, about 600% to about 900%, about 600% to about 800%, about 600% to about 700%, about 700% to about 1000%, about 700% to about 900%, about 700% to about 800%, about 800% to about 1000%, about 800% to about 900%, or about 900% to about 1000%. Expression of the gene product or protein can be measured by any method known in the art, for example, Western blot or ELISA.

[0040] In certain embodiments, the method increases the stability of a gene transcript (mRNA) encoded by a gene in a cell, tissue, or subject relative to a similar cell, tissue, or subject lacking the exogenous tRNA. For example, in certain embodiments, the methods increase mRNA stability in a cell, tissue, or subject by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000% or more as compared to a cell, tissue, or subject lacking exogenous tRNA. In certain embodiments, the method increases the stability of mRNA in a cell, tissue, or subject by about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to 60%, 20% to 40%, 40% to 200%, 40% to 180%, 40% to 160%, 40% to 140%, 40% to 120%, 40% to 100%, 40% to 80%, 40% to 60%, 60% to 200%, 60% to 180%, 60% to 160%, 60% to 1 40%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about Increase by 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200%.In certain embodiments, the method increases the stability of mRNA in a cell, tissue, or subject by about 100% to about 1000%, about 100% to about 900%, about 100% to about 800%, about 100% to about 700%, about 100% to about 600%, about 100% to about 500%, about 100% to about 400%, about 100% to about 500%, about 100% to about 600%, about 100% to about 700%, about 100% to about 800%, about 100% to about 900%, about 100% to about 10 ... 0% to about 300%, about 100% to about 200%, about 200% to about 1000%, about 200% to about 900%, about 200% to about 800%, about 200% to about 700%, about 200% to about 600%, about 200% to about 500%, about 200% to about 400%, about 200% to about 300%, about 300% to about 1000%, about 300% to about 900%, about 300% to about 800%, about 300% to 700%, 300% to 600%, 300% to 500%, 300% to 400%, 400% to 1000%, 400% to 900%, 400% to 800%, 400% to 700%, 400% to 600%, 400% to 500%, 500% to 1000%, 500% to 900%, 500% to 800% , about 500% to about 700%, about 500% to about 600%, about 600% to about 1000%, about 600% to about 900%, about 600% to about 800%, about 600% to about 700%, about 700% to about 1000%, about 700% to about 900%, about 700% to about 800%, about 800% to about 1000%, about 800% to about 900%, or about 900% to about 1000%. Stability of gene transcripts or mRNA can be measured by any method known in the art, such as Northern blot.

[0041] In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a central nervous system cell, such as a neuron.

[0042] In certain embodiments, the gene is selected from the group consisting of AGGF1, ARHGAP31, BMPR2, CHD7, COL2A1, COL3A1, CTLA4, CTNNB1, DLL4, EHMT1, ELN, ENG, FAS, FBN1, FOXG1, GATA3, GLI3, GRN, IRF6, JAG1, KCNQ4, LMX1B, MBD5, MED13L, MITF, MNX1, MYCN, NFIA, NFIX. , NOTCH1, NSD1, PAX3, PHIP, PRKAR1A, RAI1, RBPJ, RPS14, RUNX2, SALL4, SCN1A, SETBP1, SHANK3, SHH, SHOX, SLC2A1 / GLUT1, SOX10, SYNGAP1, TBX1, TBX3, TBX5, TCF4, TCOF1, TGIF1, TNXB, TRPS1, WT1, and ZIC2. In a specific embodiment, the gene is SCN1A.

[0043] In certain embodiments, the method does not substantially increase the expression of gene products from the reference gene.For example, in certain embodiments, the method increases the expression of gene products from the reference gene by less than about 5%, about 10%, about 20%, about 30%, about 40%, or about 50%.In certain embodiments, there is no detectable increase in the expression of the reference gene.In certain embodiments, the reference gene is selected from GAPDH, BAD and TRAPPC6A.In certain embodiments, the reference gene is GAPDH.

[0044] In certain embodiments, the gene is the SCN1A gene and the method increases voltage-gated sodium channel activity in a cell, tissue, or subject by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more, compared to a cell, tissue, or subject lacking exogenous tRNA. In certain embodiments, the method includes increasing voltage-gated sodium channel activity in a cell, tissue, or subject by about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 150%, about 20% to about 160%, about 20% to about 180%, about 20% to about 190%, about 20% to about 220%, about 20% to about 240%, about 20% to about 300%, about 20% to about 320%, about 20% to about 360%, about 20% to about 400%, about 20% to about 400%, about 20% to about 500%, about 20% to about 500%, about 20% to about 600%, about 20% to about 700%, about 20% to about 800%, about 20% to about 900%, about 20% to about 10 ... About 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60 % to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200%.Voltage-gated sodium channel activity can be measured by any method known in the art, for example, as described in Kalume et al. (2007) J. NEUROSCI. 27(41):11065-74, Yu et al. (2007) NAT. NEUROSCI. 9(9):1142-9, and Han et al. (2012) NATURE 489(7416):385-390.

[0045] In certain embodiments, the haploinsufficiency disorder is a disorder listed in Table 1 or Table 2 and the haploinsufficient gene is a gene listed in the corresponding row of Table 1 or Table 2. In certain embodiments, the gene is a gene listed in Table 1 or Table 2 and the first, second, and / or third non-optimal codon is a codon listed in the corresponding row of Table 1 or Table 2. In certain embodiments, the haploinsufficiency disorder is a disorder described in Table 1 or Table 2 and the first, second, and / or third non-optimal codon is a codon listed in the corresponding row of Table 1 or Table 2. [Table 1] JPEG2025010148000003.jpg255169JPEG2025010148000004.jpg248170 [Table 2] JPEG2025010148000006.jpg255170JPEG2025010148000007.jpg255168JPEG2025010148000008.jpg245170JPEG2025010148000009.jpg170170

[0046] In certain embodiments, the haploinsufficiency disorder is a disorder listed in any one of Tables 4, 5, 6, or 7 below, along with the haploinsufficient gene associated with each disorder. In certain embodiments, the gene is a gene listed in Table 4, 5, 6, or 7, and the first, second, and / or third non-optimal codon is a codon listed in the corresponding row of Table 4, 5, 6, or 7. In certain embodiments, the haploinsufficiency disorder is a disorder listed in Table 4, 5, 6, or 7, and the first, second, and / or third non-optimal codon is a codon set forth in the corresponding row of Table 4, 5, 6, or 7. Tables 4 and 5 respectively show the three and ten smallest optimal and most predominant codons of the indicated genes, calculated based on codon usage and tRNA fitness index (tAI). Tables 6 and 7 show the three and ten smallest optimal and most predominant codons for the indicated genes, respectively, calculated based on codon usage and corrected tAI based on direct quantification of tRNA levels in HEK293 cells rather than estimates of tRNA levels based on copy number of the tRNA gene.

[0047] II. Transfer RNA Decoding Nonoptimal Codons A. Transfer RNA During protein synthesis, tRNAs deliver amino acids to the ribosome for insertion into a growing protein (polypeptide) chain. tRNAs are usually about 70-100 nucleotides in length. Active tRNAs contain a 3'CCA sequence, which may be transcribed into the tRNA during synthesis or added later during post-transcriptional processing. During aminoacylation, the amino acid to be attached to a given tRNA molecule is covalently linked to the 2' or 3' hydroxyl group of the 3'-terminal ribose to form an aminoacyl-tRNA (aa-tRNA). Although amino acids can spontaneously transfer from the 2'-hydroxyl group to the 3'-hydroxyl group and vice versa, they are thought to be incorporated into the growing protein chain at the ribosome from the 3'-OH position. A loop at the other end of the folded aa-tRNA molecule contains a sequence of three bases known as the anticodon. When this anticodon sequence hybridizes or base pairs with a complementary three-base codon sequence in the ribosome-bound mRNA transcript, the aa-tRNA binds to the ribosome and incorporates that amino acid into the polypeptide chain being synthesized by the ribosome. Translation of the genetic code is influenced by tRNAs because every tRNA that base pairs with a particular codon is aminoacylated with a single specific amino acid. Each of the 61 non-terminating codons in the mRNA directs the binding of its associated aa-tRNA and the addition of a single specific amino acid to the growing protein or polypeptide chain being synthesized by the ribosome.

[0048] tRNAs are generally highly conserved and often functional across species. Thus, tRNAs derived from bacterial tRNAs, non-mammalian eukaryotic tRNAs, or mammalian (e.g., human) tRNAs can be useful in the practice of the compositions and methods described herein. Nucleotide sequences encoding naturally occurring human tRNAs are known and generally available to those of skill in the art through sources such as Genbank. See also Sprinzl et al. (2005) NUCLEIC ACIDS RES. 33: D139-40, Buckland et al. (1996) GENOMICS 35 (1): 164-71, Schimmel et al. (Eds.) (1979) "Transfer-RNA: Structure, Properties, and Recognition," Cold Spring Harbor Laboratory, Agris (1983) "The Modified Nucleosides of Transfer RNA, II," Alan R. Liss Inc. tRNAs are generally highly conserved and are often functional across species.

[0049] In certain embodiments, tRNA is or can be aminoacylated with any natural amino acid.For example, tRNA can be aminoacylated with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.In certain embodiments, the amino acid is selected from serine and arginine.

[0050] In certain embodiments, the tRNA comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0051] Sequence identity can be determined in various ways within the skill of the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.BLAST (Basic Local Alignment Search Tool) analysis (Karlin et al., (1990) PROC.NATL.ACAD.SCI.USA 87:2264-2268; Altschul, (1993) J.MOL.EVOL.36,290-300; Altschul et al., (1997) NUCLEIC ACIDS RES.25:3389-3402, incorporated by reference) using the algorithm adopted by the program blastp, blastn, blastx, tblastn and tblastx is adjusted for sequence similarity search. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. The search parameters of histogram, description, alignment, expectation (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter are at default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, which is fully incorporated by reference). The four blastn parameters can be adjusted as follows:Q=10 (gap creation penalty), R=10 (gap extension penalty), wink=1 (generates word hits at all wink.sup.th positions along the query), and gapw=16 (sets the window width within which gapped alignments are generated). Equivalent Blastp parameter settings would be Q=9, R=2, wink=1, and gapw=32. Searches may also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g., -G, cost for opening a gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for extending a gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, benefit for a nucleotide match [integer]: default = 1; -e, expectation [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, blast extension dropoff (X) to snippets: default = 20 for blastn / 7 otherwise; -X, X dropoff value for gapped alignment (snip): default = 15 for all programs, does not apply to blastn; and -Z, final X dropoff value for gapped alignment (snip): 50 for blastn, 25 otherwise). ClustalW for pairwise protein alignments may also be used (default parameters may include, for example, Blosum62 matrix and Gap Opening Penalty=10 and Gap Extension Penalty=0.1). Optimal comparisons between sequences available in the GCG package version 10.0 use DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), with equivalent settings for protein comparisons GAP=8 and LEN=2.

[0052] It is contemplated that the tRNA may contain one or more modifications. Examples of modified tRNA include acylated tRNA, alkylated tRNA, tRNA containing one or more bases other than adenine, cytosine, guanine, or uracil, tRNA covalently modified by attachment of a specific ligand or antigenic, fluorescent, affinity, reactive, spectral, or other probe moiety, tRNA containing one or more ribose moieties that are methylated or otherwise modified, aa-tRNA aminoacylated with an amino acid other than the 20 natural amino acids, including unnatural amino acids that function as reagents, carriers of specific ligands, or antigenic, fluorescent, reactive, affinity, spectral, or other probes, or any combination of these compositions. Examples of modified tRNA molecules are Soll et al. (1995) “tRNA:Structure, Biosynthesis, and Function,” ASM Press, El Yacoubi et al. (2012) ANNU.REV.GENET.46:69-95, Grosjean et al. (1998) “Modification and Editing of RNA.” ASM Press, Hendrickson et al. al.(2004)ANNU.REV.BIOCHEM.73:147-176,2004, Ibba et al.(2000)ANNU.REV.BIOCHEM.69:617-650, Johnson et al.(1995)COLD SPRING HARBOR SYMPQUANT.BIOL.60:71-82, Johnson et al. al. (1982) J.MOL.BIOL.156:113-140, Crowley et al. al. (1994) CELL 78:61-71, Beier et al. (2001) NUCLEIC ACIDS RES. 29:4767-4782, Torres et al. (2014) TRENDS MOL. MED. 20:306-314, and Bjork et al. (1987) ANNU. REV. BIOCHEM. 56:263-287.

[0053] In certain embodiments, the tRNA comprises a naturally occurring nucleotide modification. Naturally occurring tRNAs include various post-transcriptionally modified nucleotides, such as those described in Machnicka et al. (2014) RNA BIOLOGY 11(12):1619-1629, and include one or more of the residues shown in Figure 2B. In certain embodiments, the tRNA is selected from the group consisting of 2'-O-methyl guanosine or G at position 0, pseudouridine or U at position 1, 2'-O-methyl adenosine, A, 2'-O-methyl uridine, U, 2'-O-methyl cytidine, C, 2'-O-methyl guanosine, or G at position 4, N2-methyl guanosine or G at position 6, N2-methyl guanosine or G at position 7, 1-methyl adenosine, A, 1-methyl guanosine, G, or modified G at position 9, N2-methyl guanosine or G at position 10, N4-acetylcytidine or C at position 12, pseudouridine, U, 2'-O-methyl cytidine, or C at position 13, 1 1-methyladenosine, A, or modified A at position 4; dihydrouridine (D) or U at position 16; D or U at position 17; 2'-O-methylguanosine or G at position 18; 3-(3-amino-3-carboxypropyl)uridine, D, or U at position 20; 3-(3-amino-3-carboxypropyl)uridine, D, pseudouridine, U, or modified U at position 20a; D, pseudouridine, or U at position 20b; pseudouridine or U at position 25; pseudouridine, U, N2,N2-dimethylguanosine, N2-methylguanosine, G, or modified G at position 26; pseudouridine, U, N2,N2-dimethylguanosine or G, pseudouridine or U at position 28, pseudouridine or U at position 30, pseudouridine or U at position 31, 2'-O-methylpseudouridine, 2'-O-methyluridine, pseudouridine, U, 2'-O-methylcytidine, 3-methylcytidine, C or modified C at position 32, inosine, A, 2-thiouridine, 2'-O-methyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, 5-carbamoylmethyluridine, 5-carboxymethylaminomethyl-2-methyluridine, 5- ... '-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, pseudouridine, U, modified U, 2'-O-methylcytidine, 5-formyl-2'-O-methylcytidine, 5-methylcytidine, C, modified C, queuosine, mannosyl-queuosine, galactosyl-queuosine, 2'-O-methylguanosine, or G, pseudouridine or U at position 35, pseudouridine, U, or modified U at position 36, 1-methylinosine, 2-methylthio-N6-threonyl carbamide at position 37, adenosine, N6-isopentenyladenosine, N6-methyl-N6-threonylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, A, modified A, 1-methylguanosine, peroxywybutosine, wybutosine, G, or modified G, pseudouridine at position 38, U, 5-methylcytidine, C, or modified C, 1-methylpseudouridine at position 39, 2'-O-methylpseudouridine, 2'-O-methyluridine, pseudouridine, U, 2'-O-methylguanosine, or G, pseudouridine at position 40 , U, 5-methylcytidine, or C, 2'-O-methyluridine, U, or modified U at position 44, pseudouridine or U at position e11, pseudouridine or U at position e12, pseudouridine or U at position e14, 3-methylcytidine or C at position e2, 7-methylguanosine or G at position 46, D, U, or modified U at position 47, D, U, 5-methylcytidine, C, or modified C at position 48, A, modified A, 5-methylcytidine, C, or modified C at position 49, pseudouridine, U, 5-methylcytidine, or C at position 50, 5,The nucleic acid sequence includes one or more residues selected from the group consisting of 2'-O-dimethyluridine, 5-methyluridine, pseudouridine, or U, pseudouridine or U at position 55, 1-methyladenosine, A, or modified A at position 58, 2'-O-ribosyladenosine (phosphate), A, 2'-O-ribosylguanosine (phosphate), G, or modified G at position 64, pseudouridine or U at position 65, pseudouridine, U, N2-methylguanosine, or G at position 67, pseudouridine or U at position 68, and pseudouridine, U, 5-methylcytidine, or C at position 72. The numbering of the residues is based on the tRNA numbering system described in Steinberg et al., (1993) NUCLEIC ACIDS RES. 21:3011-15.

[0054] In certain embodiments, the tRNA comprises one or more nucleotide modifications selected from 5-methyluridine, pseudouridine, dihydrouridine, and 1-methyladenosine.

[0055] B. Nonoptimal codons The tRNAs useful in the compositions and methods described herein contain an anticodon that hybridizes to a non-optimal codon.

[0056] Protein synthesis is directed by a genetic code that contains 61 three-base pair codons that code for amino acids that are incorporated into the protein being synthesized, and three three-base pair codons that terminate the synthesis of the protein (called stop or termination codons). The degeneracy of the genetic code is a mathematical result based on the combination of 61 three-base pair codons that code for 20 amino acids. Synonymous codons refer to three-base pair codons that code for the same amino acid. It is generally believed that synonymous codon substitutions are silent and do not affect gene expression or the amount of the resulting functional gene product. However, synonymous codons appear to be recognized differently by the translational apparatus. This concept is called codon optimality. Conceptually, codon optimality reflects the balance between the supply of aminoacylated tRNA molecules and their demand imposed by the concentration of codons involved in translation. Thus, ribosomes quickly decode some codons because their cognate tRNAs are relatively abundant, but read others more slowly because tRNA concentrations are relatively scarce (Tuller et al. (2010) CELL 141(2):344-54; Novoa et al. (2012) TRENDS GENET. 28(11):574-81). Furthermore, the optimality of a codon is based in part on the precision of the tRNA anticodon / codon interactions, which can affect the decoding rate (Akashi (1994) GENETICS 136(3):927-35; Drummond et al. (2008) CELL 134(2):341-52).

[0057] The optimality of a codon within an mRNA transcript has been shown to affect the stability of the mRNA transcript and the rate at which the mRNA transcript is translated by ribosomes (Presnyak et al. (2015) CELL 160(6):1111-24; Radhakrishnan et al. (2016) CELL 167(1):122-132). Thus, as used herein, the term "non-optimal codon" refers to a codon in an mRNA transcript that is recognized by a tRNA that is less abundant in a cell than a different tRNA that recognizes another codon for the same amino acid. Often, the non-optimal codon is replaced with another codon that codes for the same amino acid (i.e., a synonymous codon) that is read by a more abundant tRNA in the cell, increasing the stability of the mRNA transcript and / or the rate at which the mRNA transcript is translated by ribosomes. As a result, a non-optimal codon is any codon for which overexpression of its cognate tRNA enhances mRNA stability, abundance, or translation elongation.

[0058] Codon optimality may be measured or quantified by one or more of the following approaches, including tRNA adaptation index (tAI), normalized translation efficiency (nTE), species-specific tRNA adaptation index (sTAI), codon adaptation index (CAI), gCAI and correlation coefficient of codon occurrence to mRNA stability (CSC).

[0059] tAI is an estimate of the codon optimality of each codon, taking into account the cellular concentration of tRNA (based on the copy number of tRNA gene) and an estimate of the efficiency of decoding. tAI is described, for example, in dos Reis et al. (2004) NUCLEIC ACIDS RES.32(17):5036-5044, and Mahlab et al. (2014) PLOS COMPUT.BIOL.10(1):e1003294. In certain embodiments, non-optimal codons have tAI values ​​that are smaller than the median tAI value of all codons for a given species, for example, human.

[0060] In certain embodiments, the non-optimal codons have a value of 0.47 or less as determined by tAI. For example, in certain embodiments, the non-optimal codons are CTA(Leu), TCA(Ser), CAT(His), AGT(Ser), TTA(Leu), GTA(Val), ATA(Ile), CGC(Arg), TCG(Ser), TTT(Phe), AGA(Arg), CGA(Arg), ACA(Thr), CCG(Pro), GGT(Gly), AGG(Arg), CGG(Arg), CAA(Gln), CGT(Arg), GAT(Asp), TAT(Tyr), ACC(Thr), TCC(Ser), ACG(Thr), CCC(Pro). , CTC(Leu), GTC(Val), TGG(Trp), CCA(Pro), GCG(Ala), TTG(Leu), AGC(Ser), GGA(Gly), CTG(Leu), ACT(Thr), CAC(His), TCT(Ser), GCA(Ala), CCT(Pro), CTT(Leu), GTT(Val), GGG(Gly), GAA(Glu), TTC(Phe), GAG(Glu), TGT(Cys), TAC(Tyr), GGC(Gly), AAT(Asn), GAC(Asp), and any combination thereof.

[0061] In certain embodiments, a non-optimal codon has a value of 0.25 or less, as determined by tAI. For example, in certain embodiments, the non-optimal codons are selected from CTA(Leu), TCA(Ser), CAT(His), AGT(Ser), TTA(Leu), GTA(Val), ATA(Ile), CGC(Arg), TCG(Ser), TTT(Phe), AGA(Arg), CGA(Arg), ACA(Thr), CCG(Pro), GGT(Gly), AGG(Arg), CGG(Arg), CAA(Gln), CGT(Arg), GAT(Asp), TAT(Tyr), ACC(Thr), TCC(Ser), ACG(Thr), CCC(Pro), CTC(Leu), GTC(Val), TGG(Trp), CCA(Pro), GCG(Ala), TTG(Leu), AGC(Ser), and any combination thereof.

[0062] In certain embodiments, the non-optimal codons are selected from CTA(Leu), TCA(Ser), CAT(His), AGT(Ser), TTA(Leu), GTA(Val), ATA(Ile), CGC(Arg), TCG(Ser), TTT(Phe), AGA(Arg), CGA(Arg), ACA(Thr), CCG(Pro), GGT(Gly), AGG(Arg), CGG(Arg), CAA(Gln), CGT(Arg), GAT(Asp), and any combination thereof. In certain embodiments, the non-optimal codons are selected from CTA(Leu), TCA(Ser), CAT(His), AGT(Ser), TTA(Leu), GTA(Val), ATA(Ile), CGC(Arg), TCG(Ser), TTT(Phe), and any combination thereof. In certain embodiments, the non-optimal codons are selected from CTA(Leu), TCA(Ser), CAT(His), AGT(Ser), TTA(Leu), and any combination thereof.

[0063] Codon optimality can be measured by a modified version of tAI based on direct quantification of tRNA levels in a given cell, instead of an estimate based on the copy number of the tRNA gene. tRNA levels can be determined by any method known in the art, for example, as described in Zheng et al. (2015) NAT.METHODS.12(9):835-837. For example, in certain embodiments, the non-optimal codons are selected from AGA (Arg), CTC (Leu), ATA (Ile), CCC (Pro), CTA (Leu), CTT (Leu), TAT (Tyr), CCT (Pro), TGT (Cys), CGC (Arg), GCA (Ala), TCC (Ser), TAC (Tyr), GCG (Ala), AGG (Arg), CGT (Arg), CCG (Pro), GGA (Gly), CCA (Pro), TGC (Cys), TCT (Ser), TCG (Ser), TTG (Leu), AGT (Ser), TTT (Phe), ACG (Thr), AAT (Asn), ACA (Thr), GAA (Glu), ATC (Ile), TGG (Trp), and any combination thereof. In certain embodiments, the non-optimal codons are selected from AGA(Arg), CTC(Leu), ATA(Ile), CCC(Pro), CTA(Leu), CTT(Leu), TAT(Tyr), CCT(Pro), TGT(Cys), CGC(Arg), GCA(Ala), TCC(Ser), TAC(Tyr), GCG(Ala), AGG(Arg), CGT(Arg), CCG(Pro), GGA(Gly), CCA(Pro), TGC(Cys), and any combination thereof. In certain embodiments, the non-optimal codons are selected from AGA(Arg), CTC(Leu), ATA(Ile), CCC(Pro), CTA(Leu), CTT(Leu), TAT(Tyr), CCT(Pro), TGT(Cys), CGC(Arg), and any combination thereof. In certain embodiments, the non-optimal codons are selected from AGA (Arg), CTC (Leu), ATA (Ile), CCC (Pro), CTA (Leu), and any combination thereof.

[0064] In certain embodiments, the average tAI value of codons in a gene is about 0.15 to about 0.5, about 0.15 to about 0.45, about 0.15 to about 0.4, about 0.15 to about 0.35, about 0.15 to about 0.3, about 0.15 to about 0.25, about 0.15 to about 0.2, about 0.2 to about 0.5, about 0.2 to about 0.45, about 0.2 to about 0.4, about 0.2 to about 0.35, about 0.2 to about 0.3, about 0.2 to about 0.25, Between about 0.25 to about 0.5, about 0.25 to about 0.45, about 0.25 to about 0.4, about 0.25 to about 0.35, about 0.25 to about 0.3, about 0.3 to about 0.5, about 0.3 to about 0.45, about 0.3 to about 0.4, about 0.3 to about 0.35, about 0.35 to about 0.5, about 0.35 to about 0.45, about 0.35 to about 0.4, about 0.4 to about 0.5, about 0.4 to about 0.45, and about 0.45 to about 0.5. In certain embodiments, the average tAI value of the codons in the gene is less than about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5.

[0065] stAI is an estimate of the codon optimality of each codon, taking into account the cellular concentration of tRNA and an estimate of the efficiency of decoding, weighting the efficiency of decoding in a species-specific manner. stAI is described, for example, in Sabi et al. (2014) DNA RES. 21(5):511-525. In certain embodiments, a non-optimal codon has a stAI value that is smaller than the median stAI value of all codons for a given species, e.g., human.

[0066] nTE is an estimate of the codon optimality of each codon, taking into account the cellular concentration of tRNA, the efficiency of decoding, and the estimated usage of the codon in the cellular mRNA pool. nTE is described, for example, in Pechman et al. (2013) NAT.STRUCT.MOL.BIOL.20(2):237-43. In certain embodiments, a non-optimal codon has an nTE value that is smaller than the median nTE value of all codons for a given species, for example, human.

[0067] CAI estimates the optimality of codons by scoring the codon frequency of highly expressed genes. Therefore, the optimality of codons estimated by CAI may vary depending on the gene set designated as highly expressed. CAI is described, for example, in Sharp et al. (1987) NUCLEIC ACIDS RES. 15 (3): 1281-95. gCAI is similar to CAI, but uses the entire expressed transcriptome of a given cell. In certain embodiments, non-optimal codons have a CAI value that is smaller than the median CAI value of all codons for a given species, for example, human.

[0068] CSC estimates the optimality of codons by correlating the occurrence of codons with measurements of mRNA half-life. CSC is described, for example, in Presnyak et al. (2015) CELL160(6):1111-24. In certain embodiments, non-optimal codons have a CSC value that is less than the median CSC value of all codons for a given species, for example, human. In certain embodiments, non-optimal codons have a value of 0 or less as determined by CSC.

[0069] In certain embodiments, the non-optimal codon is selected from ATA, GTA, and AGA, and any combination thereof. In certain embodiments, the non-optimal codon is ATA. In certain embodiments, the non-optimal codon is GTA. In certain embodiments, the non-optimal codon is AGA. In certain embodiments, the non-optimal codon is ATA and GTA. In certain embodiments, the non-optimal codon is ATA and AGA. In certain embodiments, the non-optimal codon is GTA and AGA. In certain embodiments, the non-optimal codon is ATA, GTA, and AGA.

[0070] In certain embodiments, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 70%, about 50% to about 60%, or about 60% to about 70% of the codons in the gene are non-optimal codons. In certain embodiments, more than about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% of the codons in the gene are non-optimal codons. In certain embodiments, more than about 40% or about 50% of the codons in the gene are non-optimal codons.

[0071] III. Methods for producing transfer RNA It is contemplated that tRNA molecules useful in practicing the compositions and methods described herein may be produced by methods known in the art, including extracellular production by synthetic chemical methods, intracellular production by recombinant DNA methods, or purified from natural sources.

[0072] For example, a DNA molecule encoding a tRNA can be synthesized chemically or by recombinant DNA methodology. For example, a sequence of a tRNA can be synthesized or cloned from a library by conventional hybridization or polymerase chain reaction (PCR) techniques using an appropriate synthetic nucleic acid primer. The resulting DNA molecule encoding a tRNA can be ligated to other appropriate nucleotide sequences, including expression control sequences, to produce, for example, a conventional gene expression construct (i.e., an expression vector) encoding the tRNA. The production of a defined gene construct is within the skill of the art. A nucleic acid encoding a desired tRNA can be incorporated (ligated) into an expression vector, such as the expression vectors described in the next section, and introduced into a host cell via conventional gene transfer or transformation techniques. Examples of host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and myeloma cells. The transformed host cell may be grown under conditions that allow the host cell to express the gene encoding the tRNA. The specific expression and purification conditions will vary depending on the expression system employed.

[0073] Alternatively, tRNA can be chemically synthesized or purified from natural sources by methods known in the art. When tRNA is aminoacylated before being introduced into a cell or administered to a subject, tRNA can be aminoacylated with a desired amino acid by any method known in the art, including chemical or enzymatic aminoacylation.

[0074] IV. Expression Vectors A tRNA of interest can be expressed in a cell of interest by incorporating a gene encoding the tRNA of interest into an expression vector. As used herein, "expression vector" refers to a vector that contains a recombinant polynucleotide that contains an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), retrotransposons (e.g., piggyback, sleeping beauty), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0075] In certain embodiments, the expression vector is a viral vector.The term "virus" is used herein to refer to any obligate intracellular parasite that does not have protein synthesis or energy generation mechanism.Examples of viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Epstein-Barr virus (EBV) vectors, polyoma viral vectors (e.g., Simian vacuolating virus 40 (SV40) vectors), pox viral vectors, and pseudotyped viral vectors.

[0076] The virus can be an RNA virus (having a genome made up of RNA) or a DNA virus (having a genome made up of DNA). In certain embodiments, the viral vector is a DNA viral vector. Exemplary DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asphalt viruses, herpes viruses (e.g., herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), papillomoviruses (e.g., HPV), polyoma viruses (e.g., simian vacuolating virus 40 (SV40)), and pox viruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus). In certain embodiments, the viral vector is an RNA virus. Examples of RNA viruses include bunyaviruses (e.g., hantaviruses), coronaviruses, Ebola viruses, flaviviruses (e.g., yellow fever virus, West Nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), polioviruses, respiratory syncytial viruses (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)), and toroviruses.

[0077] In certain embodiments, the expression vector comprises a regulatory sequence or promoter operably linked to the nucleotide sequence encoding the tRNA. The term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. The operably linked nucleotide sequences are usually contiguous. However, because enhancers generally function when separated by several kilobases from the promoter and intron sequences can be of variable length, some polynucleotide elements can be operably linked but not directly contiguous and can function transitively from different alleles or chromosomes.

[0078] The tRNA gene preferably has a strong promoter that is active in a variety of cell types. The promoter of eukaryotic tRNA genes is usually present within the structural sequence that codes for the tRNA molecule itself. Although there are elements that regulate transcriptional activity within the 5' upstream region, the length of the active transcription unit may be much shorter than 500 base pairs.

[0079] Additional examples of promoters that can be employed include, but are not limited to, retroviral LTR, SV40 promoter, human cytomegalovirus (CMV) promoter, U6 promoter, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters, including, but not limited to, histone, polIII, and β-actin promoters). Other viral promoters that can be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a promoter will be apparent to one skilled in the art from the teachings contained herein.

[0080] In certain embodiments, the expression vector comprises a tRNA coding sequence comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3, or a nucleotide sequence having 80%, 85%, 86, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3.

[0081] In certain embodiments, in addition to the tRNA coding sequence, the expression vector comprises a nucleotide sequence corresponding to a genomic DNA sequence adjacent to the corresponding wild-type tRNA gene. For example, in certain embodiments, the expression vector comprises a nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0082] Adeno-associated virus (AAV) vectors In a specific embodiment, the expression vector is an adeno-associated virus (AAV) vector.AAV is a small, non-enveloped, icosahedral virus of the genus Dependoparvovirus, family Parvoviridae.AAV has a single-stranded linear DNA genome of about 4.7 kb.AAV can infect both dividing and quiescent cells of several tissue types, and different AAV serotypes show different tissue tropism.

[0083] AAV comprises numerous serologically distinguishable types, including serotypes AAV-1 through AAV-12, as well as over 100 serotypes from non-human primates (see, e.g., Srivastava (2008) J. CELL BIOCHEM., 105(1):17-24, and Gao et al. (2004) J. VIROL., 78(12), 6381-6388). The serotype of the AAV vector used in the compositions and methods described herein can be selected by one of skill in the art based on efficiency of delivery, tissue tropism, and immunogenicity. For example, AAV-1, AAV-2, AAV-4, AAV-5, AAV-8, and AAV-9 may be used for delivery to the central nervous system; AAV-1, AAV-8, and AAV-9 may be used for delivery to the heart; AAV-2 may be used for delivery to the kidney; AAV-7, AAV-8, and AAV-9 may be used for delivery to the liver; AAV-4, AAV-5, AAV-6, AAV-9 may be used for delivery to the lungs; AAV-8 may be used for delivery to the pancreas; AAV-2, AAV-5, and AAV-8 may be used for delivery to photoreceptor cells; AAV-1, AAV-2, AAV-4, AAV-5, and AAV-8 may be used for delivery to the retinal pigment epithelium; and AAV-1, AAV-6, AAV-7, AAV-8, and AAV-9 may be used for delivery to skeletal muscle. In certain embodiments, the AAV capsid proteins include sequences disclosed in U.S. Pat. No. 7,198,951, such as, but not limited to, AAV-9 (SEQ ID NOs: 1-3 of U.S. Pat. No. 7,198,951), AAV-2 (SEQ ID NO: 4 of U.S. Pat. No. 7,198,951), AAV-1 (SEQ ID NO: 5 of U.S. Pat. No. 7,198,951), AAV-3 (SEQ ID NO: 6 of U.S. Pat. No. 7,198,951), and AAV-8 (SEQ ID NO: 7 of U.S. Pat. No. 7,198,951). AAV serotypes identified from rhesus monkeys, such as rh.8, rh.10, rh.39, rh.43, and rh.74, are also contemplated. In addition to the native AAV serotypes, modified AAV capsids have been developed to improve efficiency of delivery, tissue tropism, and immunogenicity.Examples of native and modified AAV capsids are disclosed in U.S. Pat. Nos. 7,906,111, 9,493,788, and 7,198,951, and PCT Publication No. WO2017 / 189964A2.

[0084] The wild-type AAV genome contains two 145-nucleotide inverted terminal repeats (ITRs), which contain signal sequences that direct AAV replication, genome encapsidation, and integration. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive the expression of two open reading frames encoding the rep and cap genes. The two rep promoters, coupled with differential splicing of a single AAV intron, generate four rep proteins (Rep78, Rep68, Rep52, and Rep40) from the rep gene. The Rep proteins are involved in genome replication. The Cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3), which are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

[0085] The cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, so that part or all of the 4.3 kb internal genome can be replaced with foreign DNA, for example, an expression cassette for an exogenous gene of interest. Thus, in certain embodiments, the AAV vector comprises a genome that includes an expression cassette for an exogenous gene flanked by a 5'ITR and a 3'ITR. The ITRs can be from the same serotype as the capsid or derivative. Alternatively, the ITRs can be of a different serotype than the capsid, thereby generating a pseudotype of AAV. In certain embodiments, the ITRs are from AAV-2. In certain embodiments, the ITRs are from AAV-5. At least one of the ITRs can be modified to mutate or delete the terminal separation site, thereby allowing the production of a self-complementary AAV vector.

[0086] The rep and cap proteins can be provided transitively, for example, on a plasmid, to produce AAV vectors. A host cell line that allows AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and the helper functions provided by the helper virus, such as the adenovirus genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp.1-23, 2011). Methods for producing and purifying AAV vectors have been described in detail (see, for example, Mueller et al., (2012) CURRENT PROTOCOLS IN MICROBIOLOGY, 14D.1.1-14D.1.21, Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors). Many cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, as well as insect cells (see, e.g., U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, and 8,163,543, U.S. Patent Publication No. 2002 / 0081721, and PCT Publication Nos. WO00 / 47757, WO00 / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types by one plasmid containing an ITR-flanked expression cassette and one or more additional plasmids providing additional AAV and helper virus genes.

[0087] Any serotype of AAV can be used in the compositions and methods described herein.Similarly, any adenovirus type can be used, and it is contemplated that those skilled in the art can identify the AAV and adenovirus types suitable for producing their desired recombinant AAV vector (rAAV).AAV particles can be purified, for example, by affinity chromatography, iodixonal gradient, or CsCl gradient.

[0088] AAV vectors can have single-stranded genomes larger or smaller than 4.7 kb, including oversized genomes that are 4.7 kb in size, or as large as 5.2 kb, or as small as 3.0 kb. Thus, if the exogenous gene of interest expressed from the AAV vector is small, the AAV genome can include a stuffer sequence. Furthermore, the vector genome can be substantially self-complementary, thereby allowing rapid expression in cells. In certain embodiments, the genome of a self-complementary AAV vector includes, from 5' to 3', a 5'ITR, a first nucleic acid sequence that includes a promoter and / or enhancer operably linked to the coding sequence of the gene of interest, a modified ITR that does not have a functional terminal separation site, a second nucleic acid sequence that is complementary or substantially complementary to the first nucleic acid sequence, and a 3'ITR. AAV vectors that include all types of genomes are suitable for use in the methods described herein.

[0089] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EF1α-MCS (System Bio catalog no. AAV502A-1), pAAVK-EF1α-MCS1-CMV-MCS2 (System Bio catalog number AAV503A-1), pAAV-ZsGreen1 (Clontech catalog number 6231), pAAV-MCS2 (Addgene plasmid number 46954), AAV-Stuffer (Addgene plasmid number 106248), pAAVscCBPIGpluc (Addgene plasmid number 35645), AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid number 68375), pAAV-RAM-d2TTA::TRE-MCS-WPRE-pA (Addgene plasmid number 63931), pAAV-UbC (Addgene plasmid number 62806), pAAVS These vectors include 1-P-MCS (Addgene Plasmid No. 80488), pAAV-Gateway (Addgene Plasmid No. 32671), pAAV-Puro_siKD (Addgene Plasmid No. 86695), pAAVS1-Nst-MCS (Addgene Plasmid No. 80487), pAAVS1-Nst-CAG-DEST (Addgene Plasmid No. 80489), pAAVS1-P-CAG-DEST (Addgene Plasmid No. 80490), pAAVf-EnhCB-lacZnls (Addgene Plasmid No. 35642), and pAAVS1-shRNA (Addgene Plasmid No. 82697). These vectors can be modified to be suitable for therapeutic use. For example, an exogenous gene of interest can be inserted into the multiple cloning site, and a selection marker (e.g., a gene encoding a puro or fluorescent protein) can be deleted or replaced with another (same or different) exogenous gene of interest.Further examples of AAV vectors are disclosed in U.S. Pat. Nos. 5,871,982, 6,270,996, 7,238,526, 6,943,019, 6,953,690, 9,150,882, and 8,298,818, U.S. Patent Publication No. 2009 / 0087413, and PCT Publication Nos. WO2017 / 075335A1, WO2017 / 075338A2, and WO2017 / 201258A1.

[0090] In certain embodiments, the expression vector is an AAV vector that can target the nervous system, e.g., the central nervous system, in a subject, e.g., a human subject. Exemplary AAV vectors that can target the nervous system include AAV9 variants AAV-PHP.B (see, e.g., Deverman et al. (2016) NAT.BIOTECHNOL.34(2):204-209), AAV-AS (see, e.g., Choudhury et al. (2016) MOL.THER.24:726-35), and AAV-PHP.eB (see, e.g., Chan et al. (2017) NAT.NEUROSCI.20:1172-79). Additional exemplary AAV-based strategies for targeting the nervous system are described in Bedrook et al. (2018) ANNU REV NEUROSCI.41:323-348.

[0091] Lentiviral Vectors In certain embodiments, the virus vector can be a retrovirus vector.Examples of retrovirus vectors include Moloney murine leukemia virus vector, splenic necrosis virus vector, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.Retrovirus vectors are useful as agents that mediate retrovirus-mediated gene transfer into eukaryotic cells.

[0092] In certain embodiments, the retroviral vector is a lentiviral vector.Examples of lentiviral vectors include human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV) and caprine arthritis encephalitis virus (CAEV) derived vectors.

[0093] Retroviral vectors are usually constructed such that most of the sequences coding for the structural genes of the virus are deleted and replaced with the gene of interest. Often, the structural genes (i.e., gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques known in the art. This involves digestion with an appropriate restriction endonuclease, or in some cases Bal31 exonuclease, to generate a fragment containing the appropriate portion of the packaging signal. Thus, a minimal retroviral vector contains, from 5' to 3', the 5' long terminal repeat (LTR), the packaging signal, any exogenous promoter and / or enhancer, the exogenous gene of interest, and the 3'LTR. If no exogenous promoter is provided, gene expression is driven by the 5'LTR, which is a weak promoter and requires the presence of Tat to activate expression. The structural genes can be provided in a separate vector for the manufacture of lentivirus, rendering the produced virions replication-defective. Specifically, for lentiviruses, the packaging system may include a single packaging vector encoding the Gag, Pol, Rev, and Tat genes, and a third separate vector encoding the envelope protein Env (usually VSV-G due to its broad infectivity). To improve the safety of the packaging system, the packaging vector may be split, expressing Rev from one vector and Gag and Pol from another. Tat may also be eliminated from the packaging system by using a retroviral vector containing a chimeric 5'LTR, where the U3 region of the 5'LTR is replaced with a heterologous regulatory element.

[0094] These new genes can be incorporated into the proviral backbone in several general ways. The simplest construction is one in which the structural genes of the retrovirus are replaced by a single gene that is transcribed under the control of viral regulatory sequences within the LTR. Retroviral vectors capable of introducing multiple genes into target cells have also been constructed. Typically in such vectors, one gene is under the control of the viral LTR and the second gene is expressed either separate from the spliced ​​message or under the control of its own internal promoter.

[0095] Thus, the new gene(s) are flanked by 5' and 3' LTRs that serve to facilitate transcription and polyadenylation of virion RNA, respectively. The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, which, in the context of their natural sequence, are direct repeats and include the U3, R, and U5 regions. LTRs generally provide the basic functions of retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain a number of regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region, and contains polyadenylation sequences. The R (repeat) regions flank the U3 and U5 regions. In certain embodiments, the R region contains a transactivation response (TAR) gene element, which interacts with a transactivator (tat) gene element to enhance viral replication. This element is not required in embodiments in which the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0096] In certain embodiments, the retroviral vector comprises a modified 5'LTR and / or 3'LTR. Modification of the 3'LTR is often done to improve the safety of lentiviral or retroviral systems by making the virus replication-defective. In certain embodiments, the retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication-defective retroviral vector in which the 3'LTR U3 region is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the 3'LTR U3 region is used as a template for the 5'LTR U3 region during viral replication, and no viral transcripts can be made without the U3 enhancer promoter. In further embodiments, the 3'LTR is modified such that the U5 region is replaced with, for example, an ideal polyadenylation sequence. It should be noted that modifications to the LTR, such as modifications to the 3'LTR, 5'LTR, or both 3' and 5'LTR, are also described herein.

[0097] In certain embodiments, the U3 region of 5'LTR is replaced with a heterologous promoter to drive the transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, the virus Simian Virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV), and Herpes Simplex Virus (HSV) (thymidine kinase) promoters. Typical promoters can drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility that recombination will generate replication-competent viruses, since the virus production system does not have a complete U3 sequence.

[0098] Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site). As used herein, the term "packaging signal" or "packaging sequence" refers to a sequence located in the retroviral genome that is necessary for encapsidation of retroviral RNA strands during viral particle formation (see, for example, Clever et al., 1995 J. VIROLOGY, 69(4):2101-09). The packaging signal can be a minimal packaging signal (also called psi [Ψ] sequence) that is necessary for encapsidation of the viral genome.

[0099] In certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a FLAP. As used herein, the term "FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Examples of FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al. (2000) CELL 101:173. During reverse transcription, the central initiation of positive-strand DNA at the cPPT and the central termination at the CTS leads to the formation of a triple-stranded DNA structure called the central DNA flap. Without wishing to be bound by any theory, the DNA flap may act as a cis-acting determinant of nuclear import of the lentiviral genome and / or increase the titer of the virus. In certain embodiments, the retroviral vector backbone comprises one or more FLAP elements upstream or downstream of the heterologous gene of interest in the vector. For example, in certain embodiments, the transfer plasmid comprises a FLAP element. In one embodiment, the vectors described herein contain the FLAP element isolated from HIV-1.

[0100] In certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises an export element. In one embodiment, the retroviral vector comprises one or more export elements. The term "export element" refers to a cis-acting posttranscriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) RRE, (see, e.g., Cullen et al., (1991) J. VIROL. 65:1053, and Cullen et al., (1991) CELL 58:423) and the posttranscriptional regulatory element (HPRE) of hepatitis B virus. Generally, the RNA export element is located in the 3'UTR of a gene and can be inserted as one or multiple copies.

[0101] In certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a post-transcriptional regulatory element. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acid, such as the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE, see Zufferey et al., (1999) J. VIROL., 73:2886), the post-transcriptional regulatory element (HPRE) present in Hepatitis B virus (Huang et al., MOL. CELL. BIOL., 5:3864), and the like (Liu et al., (1995), GENES DEV., 9:1766). The post-transcriptional regulatory element is generally located at the 3' end of the heterologous nucleic acid sequence. This configuration results in the synthesis of an mRNA transcript whose 5' portion comprises the heterologous nucleic acid coding sequence and whose 3' portion comprises the post-transcriptional regulatory element sequence. In certain embodiments, the vectors described herein lack or do not comprise a post-transcriptional regulatory element such as a WPRE or HPRE. Because these elements may, in some cases, increase the risk of cell transformation and / or do not substantially or significantly increase the amount of mRNA transcript or enhance mRNA stability. Thus, in certain embodiments, the vectors described herein lack or do not include a WPRE or HPRE as an additional safety measure.

[0102] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. Thus, in certain embodiments, retroviral vectors (e.g., lentiviral vectors) further comprise polyadenylation signals. The term "polyadenylation signal" or "polyadenylation sequence" as used herein refers to a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase H. Efficient polyadenylation of recombinant transcripts is desirable, since transcripts that lack polyadenylation signals are unstable and rapidly degraded. Illustrative examples of polyadenylation signals that can be used in the vectors described herein include ideal polyadenylation sequences (e.g., AATAAA, ATTAAA AGTAAA), the bovine growth hormone polyadenylation sequence (BGHpA), the rabbit β-globin polyadenylation sequence (rβgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.

[0103] In certain embodiments, retroviral vector further comprises an insulator element.The insulator element may contribute to protect therapeutic genes from integration site effects, which are mediated by cis-acting elements present in retroviral expression sequences, such as genomic DNA, and may lead to unregulated expression of transposition sequences (i.e., position effects, see, for example, Burgess-Beusse et al., (2002) PROC.NATL.ACAD.SCI., USA, 99:16433, and Zhan et al., 2001, HUM.GENET., 109:471).In certain embodiments, retroviral vector comprises an insulator element in one or both LTRs or other in the region of vector that integrates into cell genome. Examples of insulators for use in the compositions and methods described herein include, but are not limited to, chicken β-globin insulators (see Chung et al., (1993). CELL 74:505; Chung et al., (1997) PROC. NATL. ACAD. SCI., USA 94:575, and Bell et al., 1999. CELL 98:387). Examples of insulator elements include, but are not limited to, insulators from β-globin loci such as chicken HS4.

[0104] Non-limiting examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech Catalog No. 631984), pLVX-EF1alpha-IRES-mCherry (Clontech Catalog No. 631987), pLVX-Puro (Clontech Catalog No. 632159), pLVX-IRES-Puro (Clontech Catalog No. 632186), pLenti6 / V5-DEST™ (Thermo Fisher), pLenti6.2 ... Fisher), pLKO.1 (Addgene Plasmid No. 10878), pLKO.3G (Addgene Plasmid No. 14748), pSico (Addgene Plasmid No. 11578), pLJM1-EGFP (Addgene Plasmid No. 19319), FUGW (Addgene Plasmid No. 14883), pLVTHM (Addgene Plasmid No. 12247), pLVUT-tTR-KRAB (Addgene Plasmid No. 11651), pLL3.7 (Addgene Plasmid No. 11795), pLB (Addgene Plasmid No. 11619), pWPXL (Addgene Plasmid No. 12257), pWPI (Plasmid No. 12254), EF.CMV.RFP (Addgene Plasmid No. 17619), pLenti CMV Puro DEST (Addgene Plasmid No. 17452), pLenti-puro (Addgene Plasmid No. 39481), pULTRA (Addgene Plasmid No. 24129), pLX301 (Plasmid No. 25895), pHIV-EGFP (Addgene Plasmid No. 21373), pLV-mCherry (Addgene Plasmid No. 36084), pLionII (Addgene Plasmid No. 1730), pInducer10-mir-RUP-PheS (Addgene Plasmid No. 44011). These vectors can be modified to be suitable for therapeutic use.For example, the selection marker (e.g., puro, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest. Further examples of lentiviral vectors are described in U.S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, 6,313,179, and 6,411,179. Nos. 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, and 6,352,694, and in PCT Publication No. WO2017 / 091786.

[0105] Adenovirus Vectors In certain embodiments, the viral vector may be an adenoviral vector. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. The term "adenovirus" refers to any virus in the genus Adenovirus, including but not limited to the human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. Typically, adenoviral vectors are generated by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenoviral genome of an adenovirus, for example, to accommodate the insertion of a non-native nucleic acid sequence into the adenovirus for gene transfer.

[0106] Human adenoviruses can be used as the source of the adenoviral genome for adenoviral vectors. For example, the adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), or unclassified serogroups (e.g., serotypes 49 and 51), or any other adenovirus serogroup or serotype. Adenovirus serotypes 1-51 are available from the American Type Culture Collection (ATCC, Manassas, VA). Non-group C adenovirus vectors, methods of making non-group C adenovirus vectors, and methods of using non-group C adenovirus vectors are described, for example, in U.S. Patent Nos. 5,801,030, 5,837,511, and 5,849,561, and PCT Publication Nos. WO1997 / 012986 and WO1998 / 053087.

[0107] Non-human adenovirus (e.g., ape, monkey, bird, dog, sheep, or bovine adenovirus) can be used to generate adenovirus vector (i.e., as the source of the adenovirus genome of adenovirus vector). For example, adenovirus vector can be based on simian adenovirus, including monkeys from both the New World and the Old World (see, for example, Virus Taxonomy: VHIth Report of the International Committee on Taxonomy of Viruses (2005)). Phylogenetic analysis of adenoviruses that infect primates is disclosed, for example, in Roy et al. (2009) PLOS PATHOG.5(7):e1000503. Gorilla adenovirus can be used as the source of the adenovirus genome of adenovirus vector. Gorilla adenovirus and adenoviral vectors are described, for example, in PCT Publication Nos. WO2013 / 052799, WO2013 / 052811, and WO2013 / 052832. Adenoviral vectors can also contain a combination of subtypes, thereby making them "chimeric" adenoviral vectors.

[0108] Adenoviral vectors can be replication-competent, conditionally replication-competent, or replication-defective. Replication-competent adenoviral vectors can replicate in typical host cells, i.e., cells that can typically be infected by adenovirus. Conditionally replicating adenoviral vectors are adenoviral vectors that are engineered to replicate under predefined conditions. For example, replication-essential gene functions, such as those encoded by adenoviral early regions, can be operably linked to inducible, repressible, or tissue-specific transcription control sequences, such as promoters. Conditionally replicating adenoviral vectors are further described in U.S. Patent No. 5,998,205. Replication-defective adenoviral vectors are adenoviral vectors that require the complementation of one or more gene functions or regions of the adenoviral genome required for replication, for example, as a result of a defect in one or more replication-essential gene functions or regions, so that the adenoviral vector does not replicate in typical host cells, particularly in human host cells that are infected by the adenoviral vector.

[0109] In some embodiments, the adenoviral vector is replication-deficient, and therefore the replication-deficient adenoviral vector requires the complementation of at least one replication-essential gene function of one or more regions of the adenoviral genome for propagation (e.g., for forming adenoviral vector particles).The adenoviral vector can be defective in one or more replication-essential gene functions of only the early region of the adenoviral genome (i.e., E1-E4 region), only the late region of the adenoviral genome (i.e., L1-L5 region), both the early and late regions of the adenoviral genome, or all adenoviral genes (i.e., high-capacity adeno vector (HC-Ad)). See, e.g., Morsy et al. (1998) PROC. NATL. ACAD. SCI. USA 95:965-976, Chen et al. (1997) PROC. NATL. ACAD. SCI. USA 94:1645-1650, and Kochanek et al. (1999) HUM. GENE THER. 10(15):2451-9. Examples of replication-deficient adenoviral vectors are described in U.S. Pat. Nos. 5,837,511, 5,851,806, 5,994,106, 6,127,175, 6,482,616, and 7,195,896, and PCT Publication Nos. WO1994 / 028152, WO1995 / 002697, WO1995 / 016772, WO1995 / 034671, WO1996 / 022378, WO1997 / 012986, WO1997 / 021826, and WO2003 / 022311.

[0110] The replication-defective adenoviral vector described herein can be produced in a complementing cell line that provides the gene functions necessary for viral propagation at an appropriate level that are not present in the replication-defective adenoviral vector, but are necessary for generating high-titer viral vector stocks.Such complementing cell lines are known and include but are not limited to 293 cells (e.g., as described in Graham et al. (1977) J.GEN.VIROL.36:59-72), PER.C6 cells (e.g., as described in PCT Publication No. WO1997 / 000326, and U.S. Patent No. 5,994,128 and U.S. Patent No. 6,033,908), and 293-ORF6 cells (e.g., as described in PCT Publication No. WO1995 / 034671 and Brough et al. (1997) J.VIROL.71:9206-9213). Other examples of complementing cells for producing replication-defective adenoviral vectors include complementing cells generated to propagate adenoviral vectors that code for transgenes whose expression inhibits viral growth in host cells (see, for example, US Patent Publication No. 2008 / 0233650).Additional examples of complementing cells are described in US Patent Nos. 6,677,156 and 6,682,929, and PCT Publication No. WO2003 / 020879.Formulation of adenoviral vector-containing compositions is further described, for example, in US Patent Nos. 6,225,289 and 6,514,943, and PCT Publication No. WO2000 / 034444.

[0111] Additional examples of adenoviral vectors, and / or methods for making or propagating adenoviral vectors, are described in U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, 6,083,716, 6,113,913, 6,303,362, 7,067,310, and 9,073,980.

[0112] Commercially available adenoviral vector systems include the ViraPower™ Adenoviral Expression System available from Thermo Fisher Scientific, the AdEasy™ adenoviral vector system available from Agilent Technologies, and the Adeno-X™ Expression System3 available from Takara Bio USA, Inc.

[0113] Other viral vectors In certain embodiments, the viral vector can be a herpes simplex virus plasmid vector. Herpes simplex virus type 1 (HSV-1) has been demonstrated as a gene delivery vector system that may be useful for gene therapy. HSV-1 vectors have been used to transfer genes to muscle and have also been used to treat brain tumors in mice. Helper virus-dependent miniviral vectors have been developed to facilitate manipulation and allow for larger insertions (up to 140 kb). Replication-incompetent HSV amplicons have been constructed in the art. These HSV amplicons contain large deletions of the HSV genome to provide space for inserting exogenous DNA. Typically, they contain the HSV-1 packaging site, the HSV-1 "oriS" replication site, and the IE4 / 5 promoter sequence. These virions are dependent on the helper virus for propagation.

[0114] Viral vector production Methods for producing viral vectors are known in the art. Typically, the disclosed viruses are produced in suitable host cell lines using conventional techniques, including culturing transfected or infected host cells under suitable conditions to allow the production of infectious viral particles. The nucleic acids encoding viral genes and / or tRNAs can be incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques. Examples of host cells for producing the disclosed viruses include human cell lines such as HeLa, Hela-S3, HEK293, 911, A549, HER96, or PER-C6 cells. Specific production and purification conditions vary depending on the virus and production system employed.

[0115] In certain embodiments, producer cells may be administered directly to a subject, while in other embodiments, following production, infectious viral particles are harvested from the culture and optionally purified. Typical purification steps may include plaque purification, centrifugation, e.g., cesium chloride gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography or filtration steps.

[0116] IV. Pharmaceutical Compositions For therapeutic use, tRNA and / or expression vector can be combined with pharmaceutically acceptable carrier.As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problem or complication, within the scope of sound medical judgment, and that are commensurate with a reasonable benefit / risk ratio.

[0117] The term "pharmaceutically acceptable carrier" as used herein refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions, etc.), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0118] In certain embodiments, a pharmaceutical composition may include formulation materials to modify, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, the formulation materials may include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose, dextrin), proteins (such as serum albumin, gelatin, immunoglobulins), colorants, flavors and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt forming agents, and the like. counter ions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, etc.), solvents (such as glycerin, propylene glycol, polyethylene glycol, etc.), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbates, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.), stability enhancers (such as sucrose or sorbitol), tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol, etc.), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0119] In certain embodiments, the pharmaceutical composition may include nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG.TRANSL.MED. 1:10-29). In certain embodiments, the composition does not include nanoparticles or aminolipid delivery compounds, e.g., as described in U.S. Patent Publication No. 2017 / 0354672. In certain embodiments, the tRNA or expression vector introduced into the cell or administered to the subject is not conjugated or associated with another moiety, e.g., a carrier particle, e.g., an aminolipid particle. As used herein, the term "conjugate," when used in reference to two or more moieties, means that the moieties are physically associated or connected to one another, either directly or through one or more additional moieties that function as linkers, to form a structure that is sufficiently stable such that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. Typically, the moieties are attached by one or more covalent bonds or by mechanisms that include specific bonds. Alternatively, a sufficient number of weaker interactions may provide sufficient stability for the moieties to remain physically associated.

[0120] In certain embodiments, pharmaceutical compositions may include sustained or controlled delivery formulations.Techniques for formulating sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads and depot injections, are also known to those skilled in the art.Sustained release preparations may include, for example, semipermeable polymer matrices in the form of porous polymer microparticles or molded articles, such as films, or microcapsules.Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamic acid, poly(2-hydroxyethyl-inetoacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.Sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art.

[0121] The pharmaceutical composition comprising the tRNA and / or expression vector disclosed herein may be presented in dosage unit form and may be prepared by any suitable method. The pharmaceutical composition may be formulated to suit its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the tRNA and / or expression vector is administered intrathecally. In certain embodiments, the tRNA and / or expression vector is administered by injection. The formulation may be prepared by methods known in the pharmaceutical art. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components for parenteral administration include a sterile diluent such as water for injection, physiological saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose.

[0122] For intravenous administration, carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). Carriers should be stable under the conditions of manufacture and storage and protected against microorganisms. Carriers can be, for example, solvents or dispersion media including water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0123] Generally, any method of delivering nucleic acid molecules can be adapted for use with tRNA (see, for example, Akhtar et al. (1992) TRENDS CELL.) BIOL. 2(5):139-144 and PCT Publication No. WO94 / 02595). tRNA can be modified to prevent rapid degradation of tRNA by endonucleases and exonucleases in vivo, or can be delivered using a drug delivery system instead. tRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance uptake into cells and prevent degradation. tRNA molecules can also be conjugated or otherwise associated with aptamers. tRNA can also be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of tRNA molecules (negatively charged) and enhance interactions with the negatively charged cell membrane, allowing efficient uptake of tRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to RNA, such as tRNA, or can be induced to form vesicles or micelles that encase RNA (see, for example, Kim et al. (2008) JOURNAL OF CONTROLLED RELEASE 129(2):107-116). Methods for making and administering cationic RNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen et al. (2003) J.MOL.BIOL 327:761-766; Verma et al. (2003) CLIN.CANCER RES. 9:1291-1300; Arnold et al. (2007) J.HYPERTENS. 25:197-205).Some non-limiting examples of drug delivery systems useful for systemic delivery of RNA, e.g., tRNA, include DOTAP (Sorensen et al. (2003) supra; Verma et al. (2003) supra), oligofectamine, solid nucleic acid lipid particles (Zimmermann et al. (2006) NATURE 441:111-114), cardiolipin (Chien et al. (2005) CANCER GENE THER. 12:321-328; Pal et al. (2005) INT J. ONCOL. 26:1087-1091), polyethyleneimine (Bonnet et al. (2008) PHARM. RES. 25(12):2972-82; Aigner et al. (2008) PHARM. RES. 25(12):2972-82), and tRNA-binding proteins (Tan et al. (2008) PHARM. RES. 25(12):327-329). (2006) J. BIOMED. BIOTECHNOL. 71659), Arg-Gly-Asp (RGD) peptides (Liu (2006) MOL. PHARM. 3: 472-487), and polyamidoamines (Tomalia et al. (2007) BIOCHEM. SOC. TRANS. 35: 61-67, Yoo et al. (1999) PHARM. RES. 16: 1799-1804). In certain embodiments, tRNA is complexed with cyclodextrin for systemic administration. Methods and pharmaceutical compositions of RNA and cyclodextrin administration are described in U.S. Patent No. 7,427,605.

[0124] Pharmaceutical preparations are preferably sterile.Sterilization can be achieved by any suitable method, for example, filtration through a sterile filtration membrane.If the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0125] The compositions described herein can be administered locally or systemically.Administration can generally be parenteral.In some embodiments, pharmaceutical compositions are administered subcutaneously, and in even more preferred embodiments, intravenously.Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0126] Generally, a therapeutically effective amount of an active ingredient, e.g., a tRNA and / or expression vector, is in the range of 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg. In certain embodiments, a therapeutically effective amount of a viral expression vector is in the range of 10 2 ~10 15 Plaque forming units (PFU) range, e.g., 10 2 ~10 10 , 10 2 ~10 5 , 10 5 ~10 15 , 10 5 ~10 10 or 10 10 ~10 15 plaque forming units. The amount administered will depend on variables such as the type and extent of the disease or condition being treated, the patient's overall health, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage may be increased above the upper level to rapidly achieve the desired blood or tissue levels. Alternatively, the initial dosage may be less than optimal, and the daily dosage may be gradually increased over the course of treatment. Human dosage may be optimized in a conventional Phase I dose escalation study designed to run, for example, from 0.5 mg / kg to 20 mg / kg. The frequency of administration may vary depending on factors such as the route of administration, the dosage, the serum half-life, and the disease being treated. Examples of dosage frequencies are once a day, once a week, and once every two weeks. One example of a route of administration is parenteral, e.g., intravenous infusion.

[0127] V. Therapeutic Uses The compositions and methods disclosed herein can be used to treat haploinsufficiency disorders in subjects.As used herein, the term haploinsufficiency disorder refers to disorders that are mediated, strengthened, or otherwise promoted by or in association with haploinsufficiency, i.e., conditions in which a diploid subject has only a single functional copy of a gene, and the single functional copy of a gene does not produce sufficient gene product (e.g., protein) for proper gene function.As used herein, haploinsufficient gene refers to a gene that requires both alleles for proper gene function.Examples of haploinsufficiency disorders include 5q syndrome, Adams-Oliver syndrome 1, Adams-Oliver syndrome 3, Adams-Oliver syndrome 5, Adams-Oliver syndrome 6, Alagille syndrome 1, autoimmune lymphoproliferative syndrome type IA, autoimmune lymphoproliferative syndrome type V, autosomal dominant deafness-2A, brain malformations with or without urinary tract defects (BRMUTD), Carney complex type 1, CHARGE syndrome, cleidocranial dysplasia, Clarino syndrome, Dennis-Drash syndrome / Frey Jar syndrome, developmental delay, intellectual disability, obesity, and dysmorphic appearance (DIDOD), DiGeorge syndrome (TBX1-related), Dravet syndrome, Duane-radial ray syndrome, Ehlers-Danlos syndrome (classic), Ehlers-Danlos syndrome (vascular), Feingold syndrome 1, frontotemporal lobar degeneration with TDP43 inclusions (FTLD-TDP), GRN-related, GLUT1 deficiency syndrome, Greig acropolysyndactyly syndrome, hereditary hemorrhagic telangiectasia type 1, holoprosencephaly, holoprosencephaly, holoprosencephaly Prosencephaly 5, Holt-Oram syndrome, hypoparathyroidism, sensorineural deafness, and renal disease (HDR), Kleefstra syndrome 1, Klippel-Trenaunay syndrome (AAGF-associated), Leri-Weil chondrodysplasia, Marfan syndrome, mental retardation and distinctive facial features with or without heart defects (MRFACD), mental retardation, autosomal dominant 1, mental retardation, autosomal dominant 19, mental retardation, autosomal dominant 29, Nail-Patera syndrome (NPS), Phelan-McDermid syndrome, These include Pitt-Hopkins syndrome, primary pulmonary hypertension 1, Rett syndrome (congenital mutation), Smith-Magenis syndrome (RAI1-associated), Sotos syndrome 1, Sotos syndrome 2, Stickler syndrome type I, supravalvular aortic stenosis, SYNGAP1-associated intellectual disability, Treacher Collins syndrome, trichorhinophallic syndrome type I, ulnar-mammary syndrome, van der Woude syndrome 1, Waardenburg syndrome type 1, Waardenburg syndrome type 2A, and Waardenburg syndrome type 4C. Examples of haploinsufficiency disorders with associated haploinsufficient genes are shown in Tables 1 and 2.

[0128] The present disclosure provides a method for treating haploinsufficiency disorder in a subject in need of the treatment.The method includes administering an effective amount of tRNA and / or expression vector, for example, the tRNA and / or expression vector disclosed herein, alone or in combination with another therapeutic agent for treating haploinsufficiency disorder in the subject.

[0129] In certain embodiments, where the haploinsufficiency disorder is Dravet syndrome, the method reduces seizure frequency, seizure severity, and / or cognitive impairment in the subject.For example, in certain embodiments, the method reduces the seizure frequency in the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, for example, over a period of one day, one week or one month.In certain embodiments, the method reduces the seizure frequency by 50%, for example, over a period of one day, one week or one month.

[0130] As used herein, the term "effective amount" refers to an amount of an active agent (e.g., a recombinant polypeptide and / or a multimeric protein) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or doses, and is not intended to be limited to a particular formulation or route of administration.

[0131] As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., preventing its onset, and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to organisms that are treated by the methods and compositions described herein. Such organisms preferably include mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

[0132] The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. The term administered "in combination" as used herein is understood to mean that two (or more) different therapies are delivered to a subject over the course of the subject's suffering from a disorder, and at some point the effects of the therapies on the patient overlap. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, so there is an overlap in terms of administration. This is sometimes referred to herein as "concurrently" or "co-delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment than would be seen if administered in the absence of the second treatment, or a similar situation would be seen with the first treatment, or the second treatment significantly reduces symptoms. In certain embodiments, the delivery is such that the reduction in symptoms, or other parameters associated with the disorder, is greater than that observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0133] In certain embodiments, the methods or compositions described herein are administered in combination with one or more additional therapies, such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), a ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), or valproic acid. For example, during the treatment of Dravet syndrome, the methods or compositions are administered in combination with one or more additional therapies described herein, such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), a ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), or valproic acid.

[0134] Throughout the description, when compositions are described as having, including, or comprising certain components, or when processes and methods are described as having, including, or comprising certain steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0135] In this application where an element or component is said to be included in and / or selected from a list of described elements or components, it is understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0136] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether express or implied herein, may be combined in various ways without departing from the spirit and scope of the present invention. For example, when a particular compound is mentioned, that compound may be used in various embodiments of the compositions and / or methods of the present invention, unless otherwise understood from the context. In other words, within this application, the embodiments have been described and illustrated in a manner that allows for a clear and concise application to be written and drawn, but it is intended and understood that the embodiments may be combined or separated in various ways without departing from the present invention and the present invention. For example, it should be understood that all features described and depicted herein may be applied to all aspects of the invention(s) described and depicted herein.

[0137] The phrase "at least one of" should be understood to include each of the listed objects following the phrase individually, and various combinations of two or more listed objects, unless otherwise understood from context and usage. The phrase "and / or" in the context of more than two listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0138] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood as open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps unless otherwise stated or understood from the context.

[0139] When the term "about" is used before a quantitative value, unless otherwise specified, the invention also includes the specific quantitative value itself. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0140] Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0141] The use of any and all examples or exemplary language herein, such as "such as" or "including," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0142] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0143] Example 1 - tRNA expression to enhance expression of wild-type SCN1A transcript In this example, the minimal, optimal and most predominant codons in SCN1A were determined. Expression of cognate tRNAs for these codons may enhance expression of SCN1A and treat Dravet syndrome.

[0144] First, the codon usage rate of each codon in the SCN1A gene was determined. Then, the optimality of each codon in the SCN1A gene was determined using the tRNA adaptability index (tAI). tAI is an estimate of the translation efficiency of each codon that takes into account an estimate of the cellular concentration of tRNA (based on the copy number of the tRNA gene) and the efficiency of decoding, with higher tAI values ​​indicating more optimal codons. tAI is described, for example, in dos Reis et al. (2004) NUCLEIC ACIDS RES. 32(17):5036-5044, and Mahlab et al. (2014) PLOS COMPUTBIOL. 10(1):e1003294. For the calculations in this example, corrected tAI values ​​based on direct quantification of tRNA levels in HEK293 cells were used instead of estimates based on tRNA gene copy numbers (described in Zheng et al. (2015) NAT. METHODS 12(9):835-837).

[0145] The codon usage was multiplied by the inverse of the tAI value to obtain a score for each codon in the SCN1A gene. A summary of the results is shown in Table 3. In general, the highest scoring codons are the least optimal in relation to the transcriptome and are the most abundant in SCN1A, and the cognate tRNAs of these codons are the best candidates for enhancing SCN1A expression. [Table 3] JPEG2025010148000011.jpg42170

[0146] Example 2 - tRNA expression to enhance expression of wild-type SCN1A transcript In this example, the ability of ectopic delivery of selected transfer RNAs (tRNAs) to enhance expression of wild-type SCN1A in cultured cells was evaluated.

[0147] ATA (Ile), GTA (Val), and AGA (Arg) were selected as exemplary non-optimal codons for the SCN1A gene. ATAThe nucleotide sequence encoding the GTA tRNA (tRNA GTA The nucleotide sequence encoding AGA tRNA (tRNA ) is shown in SEQ ID NO:2. AGA ) is shown in SEQ ID NO:3.

[0148] The SCN1A open reading frame was amplified by RT-PCR from human brain RNA (Clontech) using primers containing a KpnI site in the forward primer and a NotI site in the reverse primer. SCN1A ORF was then cloned between the promoter and SV40 poly(A) sites of pTRE-Tight (Clontech) using the KpnI and NotI sites.

[0149] tRNA ATA , tRNA GTA , and tRNA AGA The gene sequences (including the tRNA coding sequence with 200 base pairs (bp) of flanking genomic DNA sequence) were ordered as gBlock (IDT) with an XhoI site at the 5' end and a PciI site at the 3' end. ATA The gene sequence (including the tRNA coding sequence with 200 bp of flanking genomic DNA sequence) is shown in SEQ ID NO:6. GTA The gene sequence (including the tRNA coding sequence with 200 bp of flanking genomic DNA sequence) is shown in SEQ ID NO:7. AGA The gene sequence (including the tRNA coding sequence with 200 base pairs (bp) of flanking genomic DNA sequence) is shown in SEQ ID NO:8.

[0150] All tRNA gene sequences were individually cloned into pTRE-Tight (Clontech) using the XhoI and PciI sites such that only the ampicillin resistance gene and ColE1 origin were left from pTRE-Tight. The entire tRNA gene sequence was confirmed by sequencing.

[0151] HEK293 cells were seeded at 300,000 cells per well in 6-well plates in DMEM with 10% FBS. After 24 h, each well was mock transfected (no DNA) or transfected with either 100 ng of SCN1A plasmid and 0, 100, 200, or 300 ng of each tRNA with 1, 4, 7, or 10 μL of Enhancer and 10 μL of Effectene (Qiagen). Transfected cells were incubated for 24 h and then harvested directly into Trizol (ThermoFisher) for total RNA preparation according to the manufacturer's protocol. Then, 2 μg of total RNA from each condition was treated with 4 U of TurboDNase (ThermoFisher) and purified by precipitation with 2.5 M lithium chloride. cDNA was prepared from 300 ng of DNase-treated RNA in a total reaction volume of 10 μL using SuperScript III First Strand Synthesis Supermix and random primers (ThermoFisher) according to the manufacturer's instructions. qPCR was performed in a total reaction volume of 10 μL in a Step One Real Time PCR System (Applied Biosystems) using SYBR Advantage qPCR Premix (Takara), primers for SCN1A and GAPDH, and 1 / 20th cDNA per reaction.

[0152] As shown in Figure 3, at various doses, expression of ectopic tRNA had minimal effects on the endogenous transcript, GAPDH. However, ectopic tRNA expression led to a near-linear increase in SCN1A expression, up to 7-fold at the highest dose of tRNA tested (Figure 3). No obvious toxicity to cells was observed, except at the highest concentration of tRNA.

[0153] Taken together, these results indicate that expression of an mRNA transcript, such as the SCN1A transcript, can be increased by ectopic expression of a tRNA cognate to a non-optimal codon within the transcript.

[0154] Example 3 - tRNA expression to enhance expression of haploinsufficient genes In this example, the minimal, optimal, and most overrepresented codons were determined within a gene associated with a haploinsufficiency disorder. Expression of the cognate tRNAs for these codons may enhance gene expression and treat the haploinsufficiency disorder.

[0155] The codon usage of each codon in the gene was determined as described in Example 1. The optimality of each codon in the gene was then determined using tRNA fitness index (tAI). tAI is an estimate of the translation efficiency of each codon that takes into account the estimated cellular concentration of tRNA (based on the copy number of tRNA gene) and the decoding efficiency, with higher tAI values ​​indicating more optimal codons. tAI is described, for example, in dos Reis et al. (2004) NUCLEIC ACIDS RES. 32(17): 5036-5044 and Mahlab et al. (2014) PLOS COMPUT. BIOL. 10(1): e1003294. The codon usage was multiplied by the inverse of the tAI value to obtain the score of each codon in the gene. In general, the highest scoring codons are the least optimal and most abundant in the gene in relation to the transcriptome, and the cognate tRNAs of these codons are the best candidates for enhancing gene expression.

[0156] The three smallest optimal and most prevalent codons in the indicated genes are shown in Table 4, and the ten smallest optimal and most prevalent codons in the indicated genes are shown in Table 5. [Table 4] JPEG2025010148000013.jpg141170 [Table 5] JPEG2025010148000015.jpg254170JPEG2025010148000016.jpg255170JPEG2025010148000017.jpg255170

[0157] Additionally, the minimum optimal and most overrepresented codons in the indicated genes were calculated as above (as described in Zheng et al. (2015) NAT. METHODS 12(9):835-837), except that we used modified tAI values ​​based on direct quantification of tRNA levels in HEK293 cells rather than estimates of tRNA levels based on the copy number of the tRNA gene.

[0158] The three minimal optimal and most prevalent codons for the indicated genes are shown in Table 6, and the ten minimal optimal and most prevalent codons for the indicated genes are shown in Table 7. [Table 6] JPEG2025010148000019.jpg192170 [Table 7] JPEG2025010148000021.jpg253170JPEG2025010148000022.jpg252170JPEG2025010148000023.jpg255170JPEG2025010148000024.jpg47170

[0159] Incorporation by Reference The entire disclosure of each of the patent and scientific documents referenced herein is incorporated by reference for all purposes. equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

[Claim 1] 1. A method for increasing expression in a mammalian cell of a gene product encoded by a gene comprising a first non-optimal codon, comprising: introducing into the cell an effective amount of a first exogenous tRNA, the first exogenous tRNA (i) comprising an anticodon that hybridizes to the first non-optimal codon, and (ii) capable of being aminoacylated with an amino acid, wherein introduction of the first exogenous tRNA into the cell increases the expression of the gene product compared to a similar cell lacking the first exogenous tRNA.

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