Optimized Polynucleotides for Protein Expression

Abstract

Provided herein is a method for expressing and delivering a polynucleotide that encodes a protein of interest.Specifically, the protein of interest can be a nuclease associated with a gene editing system, in which the half-life of the mRNA that encodes the engineered nuclease is increased, so that the protein level and gene editing efficiency of the engineered nuclease are increased.In particular, the mRNA comprises a specific combination of 5'UTR sequence, Kozak sequence, and 3'UTR sequence.Further provided herein are pharmaceutical compositions that include polynucleotides, and methods for modifying the genome of eukaryotic cells using the polynucleotides disclosed herein.

Description

[Technical Field]

[0001] The present invention relates to the fields of molecular biology and recombinant nucleic acid technology. In particular, the present invention relates to optimized polynucleotides useful for in vitro and in vivo protein expression, including, for example, engineered nucleases.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (P109070069WO00-SEQ-NTJ.xml, size: 183,054 bytes, created January 6, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Messenger RNA (mRNA)-based chromosome editing technology may hold the key to treating many genetic diseases. However, mRNA-based editing platforms have several areas of improvement, including the short half-life of exogenous mRNA and the associated shorter "target time" for the encoded protein to effectively edit chromosomes. Within mRNA molecules, information in the 5' and 3' untranslated regions (5' or 3' UTRs) can regulate their targeting, translation efficiency, and stability (Non-Patent Document 1). Given the wide range of regulatory effects that UTRs have on mRNAs, UTR modulation can potentially enhance both mRNA stability and translation efficiency in the system.

[0004] UTRs play an important role in the post-transcriptional regulation of gene expression. This regulation is brought about by several factors. Nucleotide motifs located in both the 5' and 3' UTRs can form secondary structures and / or directly interact with motif-specific RNA-binding proteins. In addition, UTRs can contain repeat elements that regulate expression at the RNA level. For example, CUG-binding proteins can bind to CUG repeat sequences in the 5' UTR of specific mRNAs and affect their translation efficiency (Non-Patent Document 2). Interactions between these UTR sequence elements and non-coding RNAs have also been shown to play important regulatory roles (Non-Patent Document 3). Thus, post-translational control is a combination of primary and / or secondary structure interactions with the surrounding cellular environment. In summary, UTR sequences and the cellular environment are key to RNA regulation.

[0005] mRNA turnover (i.e., mRNA half-life) is another regulatory step in protein expression. mRNAs with short half-lives do not have the opportunity to produce as much protein as mRNAs with long half-lives (regardless of 5'UTR efficiency). mRNA degradation is primarily regulated by motifs located in the 3'UTR. An example of such a motif is the AU-rich region (ARE). AREs promote mRNA degradation in response to specific intracellular and extracellular signals. AREs are classified into classes based on sequence motifs: Classes I and II are characterized by the presence of multiple copies of the AUUUA motif (Non-Patent Document 4). This ARE class controls the cytoplasmic deadenylation of mRNA by producing RNAs with short poly(A) tails of approximately 30-60 nucleotides. RNAs with such short tails are then rapidly degraded. These motifs and other similar motifs are commonly found in mRNAs encoding "fast-response" genes / proteins. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mayr,Cold Spring Harb Perspect Biol.;11(10):a034728,2019;van der Velden et al.,Int J Biochem Cell Biol.1,87-106.1999;Araujo et al.,Comp Funct Genomics;2012:475731,2012 [Non-patent document 2] Timchenko, Am J Hum Genet.64:360-364,1999 [Non-patent document 3] Sweeney et al., Proc Natl Acad Sci USA, 93:8518-8523, 1996 [Non-patent document 4] Peng et al., Mol Cell Biol.16:1490-1499,1996 Summary of the Invention [Problem to be solved by the invention]

[0007] In one aspect, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising: (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence.

[0008] In some embodiments, the 5'UTR does not include an upstream uATG sequence or an upstream open reading frame sequence.

[0009] In some embodiments, the 5'UTR further comprises a eukaryotic initiation factor (eIF) recruitment sequence. In some embodiments, the eIF recruitment sequence comprises an eIF4A recruitment sequence. In some embodiments, the eIF recruitment sequence comprises an eIF4G recruitment sequence. In some embodiments, the eIF4G recruitment sequence comprises an APT17 sequence. In some embodiments, the APT17 sequence comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence set forth in SEQ ID NO: 14. In some embodiments, the APT17 sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 14.

[0010] In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing a heterologous protein start codon. In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing a heterologous protein start codon with a change in free energy (ΔG) of less than about -10 kcal / mol to about -80 kcal / mol. In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing a heterologous protein start codon with a change in free energy (ΔG) of less than about -30 kcal / mol to about -50 kcal / mol. In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing a heterologous protein start codon with a change in free energy (ΔG) of less than about -30 kcal / mol. In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing a heterologous protein start codon with a change in free energy (ΔG) of less than about -50 kcal / mol.

[0011] In some embodiments, the 5'UTR further comprises a UTR Kozak sequence. In some embodiments, the UTR Kozak sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149. In some embodiments, the UTR Kozak sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 114.

[0012] In some embodiments, the 5'UTR is about 30 to about 250 nucleotides in length.

[0013] In some embodiments, the 5'UTR further comprises an internal ribosome entry site (IRES).

[0014] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-7.

[0015] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:1.

[0016] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence set forth in SEQ ID NO:2.

[0017] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:3.

[0018] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence set forth in SEQ ID NO:4.

[0019] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence set forth in SEQ ID NO:5.

[0020] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:6.

[0021] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence set forth in SEQ ID NO:7.

[0022] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-7. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7.

[0023] In some embodiments, the 3'UTR has less than about 5 AU-rich regions (AREs). In some embodiments, the 3'UTR has less than about 3 AREs. In some embodiments, the 3'UTR does not contain any AREs. In some embodiments, the AREs are class I AREs. In some embodiments, the AREs are class II AREs. In some embodiments, the AREs are class III AREs.

[0024] In some embodiments, the 3'UTR is about 30 to about 700 nucleotides in length. In some embodiments, the 3'UTR is about 100 to about 500 nucleotides in length. In some embodiments, the 3'UTR is about 50 to about 250 nucleotides in length.

[0025] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence set forth in any one of SEQ ID NOs:8-13.

[0026] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:8.

[0027] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:9.

[0028] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:10.

[0029] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:11.

[0030] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:12.

[0031] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:13.

[0032] In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 8-13. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 13.

[0033] In some embodiments, the polynucleotide further comprises a modification to the coding sequence of the heterologous protein to reduce ribosomal stacking or stalling during protein translation of the coding sequence, the modification comprising changing one or more three-base codons in the coding sequence that promote ribosomal stalling to three-base codons that reduce ribosomal stalling, thereby reducing ribosomal stalling or stacking during protein translation of the heterologous protein. In some embodiments, the modification does not change the amino acid sequence of the heterologous protein. In some embodiments, the modification comprises modifying codons encoding amino acid positions 3, 4, 5, 6, 7, 8, 9, or 10 of the coding sequence. In some embodiments, the modification comprises modifying codons encoding amino acid positions 3, 4, and 5 of the coding sequence.

[0034] In some embodiments, the polynucleotide further comprises a modification to the coding sequence of the heterologous protein to reduce the thymidine or uridine content of the coding sequence, where the modification does not change the amino acid sequence of the heterologous protein. In some embodiments, the modification comprises changing a first three-base codon containing thymidine or uridine that encodes an amino acid to an alternative three-base codon that contains less thymidine or uridine than the first three-base codon. In some embodiments, the modification comprises changing a first three-base codon containing thymidine or uridine that encodes an amino acid to an alternative three-base codon that does not contain thymidine or uridine. In some embodiments, the coding sequence has a thymidine or uridine content that is reduced by 10% to 90% compared to a coding sequence that has not been modified to reduce the thymidine or uridine content. In some embodiments, the coding sequence has a thymidine or uridine content that is reduced by 30% to 70% compared to a coding sequence that has not been modified to reduce the thymidine or uridine content. In some embodiments, the coding sequence has a thymidine or uridine content that is reduced by about 40% compared to a coding sequence that has not been modified to reduce the thymidine or uridine content.

[0035] In some embodiments, the polynucleotide further comprises a modification to the coding sequence of the heterologous protein to increase the guanosine or cytosine content of the coding sequence, where the modification does not change the amino acid sequence of the heterologous protein. In some embodiments, the modification comprises changing the first three-base codon for uridine that encodes the amino acid to an alternative three-base codon with an increased guanosine or cytosine content. In some embodiments, the coding sequence has a 10% to 50% increased guanosine or cytosine content compared to a coding sequence that has not been modified to increase the guanosine or cytosine content.

[0036] In some embodiments, the nucleic acid sequence comprises a promoter operably linked to the nucleic acid sequence encoding the heterologous protein.

[0037] In some embodiments, the heterologous protein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the heterologous protein. In some embodiments, the NLS is located at the C-terminus of the heterologous protein. In some embodiments, the heterologous protein comprises a first NLS at the N-terminus and a second NLS at the C-terminus of the heterologous protein. In some embodiments, the first NLS and the second NLS are identical. In some embodiments, the first NLS and the second NLS are not identical. In some embodiments, the NLS comprises an SV40 NLS, a CMYC NLS, or an NLS5 NLS. In some embodiments, the NLS comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in any one of SEQ ID NOs: 15-18. In some embodiments, the NLS comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-18.

[0038] In some embodiments, the heterologous protein is an engineered nuclease. In some embodiments, the engineered nuclease is an engineered meganuclease, TALEN, zinc finger nuclease, CRISPR system nuclease, compact TALEN, or megaTAL.

[0039] In some embodiments, the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region, and wherein the first subunit and the second subunit each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 169. In some embodiments, the first subunit and the second subunit each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 169. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 170. In some such embodiments, the codons encoding amino acids that are conserved between the first subunit and the second subunit are wobble, i.e., are not identical to each other, but still encode the same amino acids.

[0040] In some embodiments, the 5' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 7, and the 3' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7, and the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the 5' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 1, and the 3' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 1, and the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10.

[0041] In some embodiments, the 5' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 2, and the 3' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 2, and the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10.

[0042] In some embodiments, the 5' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 4, and the 3' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 4, and the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10.

[0043] In some embodiments, the 5' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 7, and the 3' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7, and the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10.

[0044] In some embodiments, the 5' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 7, and the 3' UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the 5' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7, and the 3' UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 8.

[0045] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:7; the 5'UTR comprises a UTR Kozak sequence comprising a nucleic acid sequence set forth in any one of SEQ ID NOs:50-149; the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence; the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease; the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% sequence identity to the sequence set forth in SEQ ID NO: 15; the coding sequence for the heterologous protein is modified to have a reduced thymidine or uridine content; the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 9; and the 3'UTR does not contain any AREs.

[0046] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7, the 5'UTR comprises a UTR Kozak sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149, the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% sequence identity to SEQ ID NO: 15, the coding sequence of the heterologous protein is modified to have reduced thymidine or uridine content, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 9, and the 3'UTR does not comprise any AREs.

[0047] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:1; the 5'UTR comprises a UTR Kozak sequence comprising a nucleic acid sequence set forth in any one of SEQ ID NOs:50-149; the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence; and the heterologous protein comprises a first nucleotide sequence at the N-terminus of the engineered nuclease. an engineered nuclease comprising a first NLS and a second NLS at its C-terminus, wherein the first and second NLSs are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15; wherein the coding sequence for the heterologous protein is modified to have a reduced thymidine or uridine content; and wherein the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10; and wherein the 3'UTR does not contain any AREs.

[0048] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 1, the 5'UTR comprises a UTR Kozak sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149, the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15, the coding sequence of the heterologous protein is modified to have reduced thymidine or uridine content, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10, and the 3'UTR does not comprise any AREs.

[0049] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:2; the 5'UTR comprises a UTR Kozak sequence comprising a nucleic acid sequence set forth in any one of SEQ ID NOs:50-149; the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence; the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease; the first and second NLSs are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15; the coding sequence of the heterologous protein is modified to have a reduced thymidine or uridine content; the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10; and the 3'UTR does not contain any AREs. In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 2, the 5'UTR comprises a UTR Kozak sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149, the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15, the coding sequence of the heterologous protein is modified to have reduced thymidine or uridine content, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10, and the 3'UTR does not comprise any AREs.

[0050] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:4; the 5'UTR comprises a UTR Kozak sequence comprising a nucleic acid sequence set forth in any one of SEQ ID NOs:50-149; the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence; and the heterologous protein comprises a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease. wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15; the coding sequence for the heterologous protein is modified to have reduced thymidine or uridine content; and the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10; and the 3'UTR does not contain any AREs.

[0051] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 4, the 5'UTR comprises a UTR Kozak sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149, the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15, the coding sequence of the heterologous protein is modified to have reduced thymidine or uridine content, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10, and the 3'UTR does not comprise any AREs.

[0052] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:7; the 5'UTR comprises a UTR Kozak sequence comprising a nucleic acid sequence set forth in any one of SEQ ID NOs:50-149; the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence; and the heterologous protein comprises a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease. wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15; the coding sequence for the heterologous protein is modified to have reduced thymidine or uridine content; and the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO: 10; and the 3'UTR does not contain any AREs.

[0053] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7, the 5'UTR comprises a UTR Kozak sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149, the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 15, the coding sequence of the heterologous protein is modified to have reduced thymidine or uridine content, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 10, and the 3'UTR does not comprise any AREs.

[0054] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:7; the 5'UTR comprises a UTR Kozak sequence comprising a nucleic acid sequence set forth in any one of SEQ ID NOs:50-149; the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence; and the heterologous protein comprises a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease. wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO:15; the coding sequence for the heterologous protein is modified to have reduced thymidine or uridine content; and the 3'UTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence set forth in SEQ ID NO:8; and the 3'UTR does not contain any AREs.

[0055] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 7, the 5'UTR comprises a UTR Kozak sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 50-149, the 5'UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15, the coding sequence of the heterologous protein is modified to have reduced thymidine or uridine content, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 8, and the 3'UTR does not comprise any AREs.

[0056] In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the 5' cap comprises a 5' methylguanosine cap. In some embodiments, the uridine present in the mRNA is pseudouridine or 2-thiouridine. In some embodiments, the uridine present in the mRNA is methylated. In some embodiments, the uridine present in the mRNA is N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0057] In another aspect, the present disclosure provides a recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, wherein the nucleic acid sequence comprises (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, and the polynucleotide is a polynucleotide described herein. In some embodiments, the recombinant DNA construct encodes a recombinant virus comprising the polynucleotide. In some embodiments, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the heterologous protein.

[0058] In another aspect, the present disclosure provides a recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, wherein the nucleic acid sequence comprises (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, and the polynucleotide is a polynucleotide described herein. In some embodiments, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the heterologous protein.

[0059] In another aspect, the present disclosure provides a lipid nanoparticle composition comprising a lipid nanoparticle comprising a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, wherein the polynucleotide is a polynucleotide described herein. In some embodiments, the polynucleotide included in the lipid nanoparticle composition is an mRNA described herein.

[0060] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, wherein the nucleic acid sequence comprises (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, and wherein the polynucleotide is a polynucleotide described herein.

[0061] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant DNA construct described herein.

[0062] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant virus described herein.

[0063] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle composition described herein.

[0064] In another aspect, the disclosure provides a eukaryotic cell comprising a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, wherein the polynucleotide is a polynucleotide described herein.

[0065] In another aspect, the disclosure provides a method for expressing a heterologous protein in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, wherein the polynucleotide is a polynucleotide described herein, and wherein the heterologous protein is expressed in the eukaryotic cell.

[0066] In some embodiments of the method for expressing a heterologous protein in a eukaryotic cell, the protein level of the heterologous protein is increased in the eukaryotic cell compared to a control eukaryotic cell of the same type, and the heterologous protein is introduced into the control eukaryotic cell by a control polynucleotide comprising a nucleic acid sequence encoding the heterologous protein, and the control polynucleotide does not comprise a 5' UTR or a 3' UTR. In some embodiments, mRNA persists longer in the eukaryotic cell compared to a control eukaryotic cell of the same type, and the control polynucleotide is introduced into the control eukaryotic cell, and the control polynucleotide is mRNA, and the control polynucleotide does not comprise a 5' UTR or a 3' UTR. In some embodiments of the method for expressing a heterologous protein in a eukaryotic cell, the control polynucleotide does not comprise a 5' UTR. In some embodiments, the control polynucleotide does not comprise a 3' UTR. In some embodiments, the control polynucleotide does not comprise a 5' or 3' UTR. In some embodiments, the control polynucleotide does not comprise a 5' UTR described herein. In some embodiments, the control polynucleotide does not comprise a 3' UTR described herein. In some embodiments, the control polynucleotide does not comprise the 5' and 3' UTRs described herein. In some embodiments, the control polynucleotide does not comprise the modifications of the polynucleotide described herein. In some embodiments, the control polynucleotide does not comprise a nucleic acid sequence comprising a coding sequence encoding a heterologous protein comprising an NLS described herein.

[0067] In some embodiments of the method for expressing a heterologous protein in a eukaryotic cell, the control polynucleotide does not contain pseudouridine or 2-thiouridine. In some embodiments, the control polynucleotide is unmethylated. In some embodiments, the control polynucleotide does not contain N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0068] In some embodiments of the methods of expressing a heterologous protein in a eukaryotic cell, protein levels are increased by about 2-10 fold in the eukaryotic cell compared to a control eukaryotic cell. In some embodiments, mRNA persistence is increased by about 2-10 fold in the eukaryotic cell compared to a control eukaryotic cell. In some embodiments, the mRNA remains in the cell for about 1 hour to about 96 hours. In some embodiments, the mRNA remains in the cell for about 8 hours to about 48 hours. In some embodiments, the mRNA remains in the cell for at least 8 hours. In some embodiments, the mRNA remains in the cell for at least 24 hours.

[0069] In some embodiments of the methods of expressing a heterologous protein in a eukaryotic cell, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is part of a tissue. In some embodiments, the eukaryotic cell is in a mammal. In some embodiments, the eukaryotic cell is in a human.

[0070] In some embodiments of the method of expressing a heterologous protein in a eukaryotic cell, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is a recombinant DNA construct described herein. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus described herein.

[0071] In another aspect, the disclosure provides a method for expressing a heterologous protein in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, wherein the polynucleotide is a polynucleotide described herein, and wherein the heterologous protein is expressed in the eukaryotic cell.

[0072] In some embodiments of the methods for expressing a heterologous protein in a eukaryotic cell, the protein level of the heterologous protein is increased in the eukaryotic cell compared to a control eukaryotic cell of the same type, and the heterologous protein is introduced into the control eukaryotic cell by a control polynucleotide comprising a nucleic acid sequence encoding the heterologous protein. In some embodiments, mRNA persists longer in the eukaryotic cell compared to the control eukaryotic cell of the same type, and the control polynucleotide is introduced into the control eukaryotic cell, and the control polynucleotide is mRNA.

[0073] In some embodiments of the method for expressing a heterologous protein in a eukaryotic cell, the protein level of the heterologous protein is reduced in the eukaryotic cell compared to a control eukaryotic cell of the same type, and the heterologous protein is introduced into the control eukaryotic cell by a control polynucleotide comprising a nucleic acid sequence encoding the heterologous protein. In some embodiments, the mRNA remains shorter in the eukaryotic cell compared to the control eukaryotic cell of the same type, and the control polynucleotide is introduced into the control eukaryotic cell, and the control polynucleotide is mRNA.

[0074] In some embodiments, the protein level of the heterologous protein is reduced when the 5'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In some embodiments, when the 5'UTR comprises the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and comprises the 3'UTR of the HBA2 gene (SEQ ID NO: 8), the protein level of the heterologous protein is reduced. In some embodiments, the protein level of the heterologous protein is reduced when the 5'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the XBG gene (SEQ ID NO: 12). In some embodiments, when the 5'UTR comprises the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR of the XBG gene (SEQ ID NO: 12), the protein level of the heterologous protein is reduced. In some embodiments, the persistence of an mRNA encoding a heterologous protein is reduced when the 5'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In some embodiments, when the 5'UTR comprises the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR of the HBA2 gene (SEQ ID NO: 8), the persistence of the mRNA encoding the heterologous protein is reduced.

[0075] In some embodiments, the persistence of an mRNA encoding a heterologous protein is reduced when the 5'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the XBG gene (SEQ ID NO: 12). In some embodiments, when the 5'UTR comprises the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and the 3'UTR of the XBG gene (SEQ ID NO: 12), the persistence of the mRNA encoding the heterologous protein is reduced.

[0076] In some embodiments of the methods for expressing a heterologous protein in a eukaryotic cell, the control polynucleotide described herein comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13). In some particular embodiments of the methods for expressing heterologous proteins in eukaryotic cells, the control polynucleotide described herein comprises a 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0077] In some embodiments of the method for expressing a heterologous protein in a eukaryotic cell, the control polynucleotide does not comprise a 5' UTR. In some embodiments, the control polynucleotide does not comprise a 3' UTR. In some embodiments, the control polynucleotide does not comprise a 5' and a 3' UTR. In some embodiments, the control polynucleotide does not comprise a 5' UTR described herein. In some embodiments, the control polynucleotide does not comprise a 3' UTR described herein. In some embodiments, the control polynucleotide does not comprise a 5' and a 3' UTR described herein. In some embodiments, the control polynucleotide does not comprise a polynucleotide modification described herein. In some embodiments, the control polynucleotide does not comprise a nucleic acid sequence comprising a coding sequence encoding a heterologous protein comprising an NLS described herein.

[0078] In some embodiments of the method for expressing a heterologous protein in a eukaryotic cell, the control polynucleotide does not contain pseudouridine or 2-thiouridine. In some embodiments, the control polynucleotide is unmethylated. In some embodiments, the control polynucleotide does not contain N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0079] In some embodiments of the methods of expressing a heterologous protein in a eukaryotic cell, protein levels are increased by about 2-10 fold in the eukaryotic cell compared to a control eukaryotic cell. In some embodiments, mRNA persistence is increased by about 2-10 fold in the eukaryotic cell compared to a control eukaryotic cell. In some embodiments, the mRNA remains in the cell for about 1 hour to about 96 hours. In some embodiments, the mRNA remains in the cell for about 8 hours to about 48 hours. In some embodiments, the mRNA remains in the cell for at least 8 hours. In some embodiments, the mRNA remains in the cell for at least 24 hours.

[0080] In some embodiments of the methods of expressing a heterologous protein in a eukaryotic cell, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is part of a tissue. In some embodiments, the eukaryotic cell is in a mammal. In some embodiments, the eukaryotic cell is in a human.

[0081] In some embodiments of the method of expressing a heterologous protein in a eukaryotic cell, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is a recombinant DNA construct described herein. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus described herein.

[0082] In another aspect, the disclosure provides a method of making a genetically engineered eukaryotic cell comprising a modified genome of a eukaryotic cell, the method comprising introducing into the eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, wherein the polynucleotide is a polynucleotide described herein, and the heterologous protein is an engineered nuclease, wherein the engineered nuclease is expressed in the eukaryotic cell and generates a cleavage site in the genome at the engineered nuclease recognition sequence, resulting in a modified genome in the eukaryotic cell.

[0083] In some embodiments of the methods for producing a genetically engineered eukaryotic cell comprising a modified genome, the protein level of the engineered nuclease is increased in the eukaryotic cell compared to a control eukaryotic cell of the same type, and the engineered nuclease is introduced into the control eukaryotic cell by a control polynucleotide comprising a nucleic acid sequence encoding the engineered nuclease, wherein the control polynucleotide does not comprise a 5' UTR or a 3' UTR. In some embodiments, mRNA persists longer in the eukaryotic cell compared to a control eukaryotic cell of the same type, and the control polynucleotide is introduced into the control eukaryotic cell, wherein the control polynucleotide is mRNA, and wherein the control polynucleotide does not comprise a 5' UTR or a 3' UTR.

[0084] In some embodiments, the engineered nuclease is an engineered meganuclease, a TALEN, a zinc finger nuclease, a CRISPR system nuclease, a compact TALEN, or a megaTAL.

[0085] In some embodiments, the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region, and wherein the first subunit and the second subunit each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 169. In some embodiments, the first subunit and the second subunit each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 169. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 170. In some such embodiments, the codons encoding amino acids that are conserved between the first subunit and the second subunit are wobble, i.e., are not identical to each other, but still encode the same amino acids.

[0086] In some embodiments of the methods of generating a genetically engineered eukaryotic cell comprising a modified genome, the control polynucleotide does not comprise a 5' UTR. In some embodiments, the control polynucleotide does not comprise a 3' UTR. In some embodiments, the control polynucleotide does not comprise a 5' and a 3' UTR. In some embodiments, the control polynucleotide does not comprise a 5' UTR described herein. In some embodiments, the control polynucleotide does not comprise a 3' UTR described herein. In some embodiments, the control polynucleotide does not comprise a 5' and a 3' UTR described herein. In some embodiments, the control polynucleotide does not comprise a polynucleotide modification described herein.

[0087] In some embodiments of the method for producing a genetically engineered eukaryotic cell comprising a modified genome, the control polynucleotide does not comprise pseudouridine or 2-thiouridine. In some embodiments, the control polynucleotide is unmethylated. In some embodiments, the control polynucleotide does not comprise N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0088] In some embodiments of the methods of producing a genetically engineered eukaryotic cell comprising a modified genome, protein levels are increased by about 2-10 fold in the eukaryotic cell compared to a control eukaryotic cell. In some embodiments, mRNA persistence is increased by about 2-10 fold in the eukaryotic cell compared to a control eukaryotic cell. In some embodiments, the mRNA remains in the cell for about 1 hour to about 96 hours. In some embodiments, the mRNA remains in the cell for about 8 hours to about 48 hours. In some embodiments, the mRNA remains in the cell for at least 8 hours. In some embodiments, the mRNA remains in the cell for at least 24 hours.

[0089] In some embodiments of the methods of generating a genetically engineered eukaryotic cell comprising a modified genome, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is part of a tissue. In some embodiments, the eukaryotic cell is in a mammal. In some embodiments, the eukaryotic cell is in a human. In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is a recombinant DNA construct described herein. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus described herein.

[0090] In another aspect, the disclosure provides a method of treating a disease in a subject, the method comprising administering a therapeutically effective amount of a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising: (a) a 5' untranslated region (UTR), (b) a coding sequence encoding the heterologous protein, (c) a 3' UTR, and (d) a polyA sequence, wherein the polynucleotide is a polynucleotide described herein, and the heterologous protein is a therapeutic protein.

[0091] In some embodiments of the treatment methods, the protein level of a heterologous protein is increased in a subject compared to a control subject, and the heterologous protein is introduced into the control subject by a control polynucleotide comprising a nucleic acid sequence encoding the heterologous protein, wherein the control polynucleotide does not comprise a 5'UTR or a 3'UTR. In some embodiments, mRNA remains longer in the subject compared to the control subject, and a control polynucleotide is introduced into the control subject, and the control polynucleotide is mRNA, and the control polynucleotide does not comprise a 5'UTR or a 3'UTR. In some embodiments, the control polynucleotide does not comprise a 5'UTR. In some embodiments, the control polynucleotide does not comprise a 3'UTR. In some embodiments, the control polynucleotide does not comprise a 5'UTR or a 3'UTR. In some embodiments, the control polynucleotide does not comprise a 5'UTR described herein. In some embodiments, the control polynucleotide does not comprise a 3'UTR described herein. In some embodiments, the control polynucleotide does not comprise a 5'UTR or a 3'UTR described herein.

[0092] In some embodiments of the methods of treatment, the control polynucleotide does not comprise a modification of a polynucleotide described herein, hi some embodiments, the control polynucleotide does not comprise a nucleic acid sequence comprising a coding sequence encoding a heterologous protein comprising an NLS described herein.

[0093] In some embodiments, the control polynucleotide does not contain a nucleic acid sequence comprising a coding sequence encoding a heterologous protein comprising an NLS described herein. In some embodiments of the methods of treatment, the control polynucleotide does not contain pseudouridine or 2-thiouridine. In some embodiments, the control polynucleotide is unmethylated. In some embodiments, the control polynucleotide does not contain N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine. In some embodiments of the methods of treatment, protein levels are increased by about 2-10 fold in the subject compared to a control subject. In some embodiments, mRNA persistence is improved by about 2-10 fold in the subject compared to a control subject.

[0094] In some embodiments of the treatment method, the control polynucleotide does not contain pseudouridine or 2-thiouridine. In some embodiments, the control polynucleotide is unmethylated. In some embodiments, the control polynucleotide does not contain N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0095] In some embodiments of the methods of treatment, protein levels are increased by about 2-10 fold in the subject compared to a control subject. In some embodiments, mRNA persistence is increased by about 2-10 fold in the subject compared to a control subject. In some embodiments, the mRNA remains intracellularly for about 1 hour to about 96 hours. In some embodiments, the mRNA remains intracellularly for about 8 hours to about 48 hours. In some embodiments, the mRNA remains intracellularly for at least 8 hours. In some embodiments, the mRNA remains intracellularly for at least 24 hours.

[0096] In some embodiments of the treatment method, therapeutic protein is a peptide or protein as part of vaccine, antibody, engineered nuclease, RNA modifying enzyme or DNA modifying enzyme.In some embodiments, therapeutic protein is engineered nuclease.In some embodiments, engineered nuclease is engineered meganuclease, TALEN, zinc finger nuclease, CRISPR system nuclease, compact TALEN or megaTAL.

[0097] In some embodiments, the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region, and wherein the first subunit and the second subunit each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 169. In some embodiments, the first subunit and the second subunit each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 169. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 170. In some such embodiments, the codons encoding amino acids that are conserved between the first subunit and the second subunit are wobble, i.e., are not identical to each other, but still encode the same amino acids.

[0098] In some embodiments of the method of treatment, the polynucleotide is mRNA. In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is a recombinant DNA construct described herein. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus described herein. In some embodiments, the polynucleotide is administered by a pharmaceutical composition described herein.

[0099] A brief description of arrays

[0100] SEQ ID NO: 1 shows the DNA nucleic acid sequence of the 5'ALB UTR.

[0101] SEQ ID NO: 2 shows the DNA nucleic acid sequence of the 5'FGA UTR.

[0102] SEQ ID NO: 3 shows the DNA nucleic acid sequence of the 5'FTH1 UTR.

[0103] SEQ ID NO: 4 shows the DNA nucleic acid sequence of the 5'GAPDH UTR.

[0104] SEQ ID NO: 5 shows the DNA nucleic acid sequence of the 5'HBA2 UTR.

[0105] SEQ ID NO: 6 shows the DNA nucleic acid sequence of the 5'SNRPB variant 1 UTR.

[0106] SEQ ID NO: 7 shows the DNA nucleic acid sequence of the 5'XBG UTR.

[0107] SEQ ID NO: 8 shows the DNA nucleic acid sequence of the 3'HBA2 UTR.

[0108] SEQ ID NO: 9 shows the DNA nucleic acid sequence of the 3'HBB UTR.

[0109] SEQ ID NO: 10 shows the DNA nucleic acid sequence of the 3'SNRPB variant 1 UTR.

[0110] SEQ ID NO: 11 shows the DNA nucleic acid sequence of the 3'SNRPB variant 2 UTR.

[0111] SEQ ID NO: 12 shows the DNA nucleic acid sequence of the 3'XBG UTR.

[0112] SEQ ID NO: 13 shows the DNA nucleic acid sequence of the 3'WPRE UTR.

[0113] SEQ ID NO: 14 shows the DNA nucleic acid sequence of the APT17 recruitment sequence.

[0114] SEQ ID NO: 15 shows the amino acid sequence of the SV40 nuclear localization sequence.

[0115] SEQ ID NO: 16 shows the amino acid sequence of the NLS5 nuclear localization sequence.

[0116] SEQ ID NO: 17 shows the amino acid sequence of the CMYC nuclear localization sequence.

[0117] SEQ ID NO: 18 shows the amino acid sequence of the SV40H2 nuclear localization sequence.

[0118] SEQ ID NO: 19 shows the DNA nucleic acid sequence of the SV40 nuclear localization sequence.

[0119] SEQ ID NO: 20 shows the DNA nucleic acid sequence of the NLS5 nuclear localization sequence.

[0120] SEQ ID NO: 21 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the APT17 ribosome recruitment sequence, the 5'HBA2 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'WPRE UTR.

[0121] SEQ ID NO: 22 shows a DNA nucleic acid sequence comprising from 5' to 3' the coding sequence for the T7AG promoter, the APT17 ribosome recruitment sequence, the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'WPRE UTR.

[0122] SEQ ID NO: 23 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the coding sequence for the NLS5 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'XBG UTR.

[0123] SEQ ID NO: 24 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'XBG UTR.

[0124] SEQ ID NO: 25 shows a DNA nucleic acid sequence comprising, from 5' to 3', the T7AG promoter, the 5'SNRPB V1 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'SNRPB V1 UTR.

[0125] SEQ ID NO: 26 shows a DNA nucleic acid sequence comprising, from 5' to 3', the T7AG promoter, the 5'SNRPB V1 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'SNRPB V2 UTR.

[0126] SEQ ID NO: 27 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'WPRE UTR.

[0127] SEQ ID NO: 28 shows a DNA nucleic acid sequence comprising, from 5' to 3', the T7AG promoter, the 5'HBA2 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'HBA2 UTR.

[0128] SEQ ID NO: 29 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'HBB UTR.

[0129] SEQ ID NO: 30 shows a DNA nucleic acid sequence comprising, from 5' to 3', a T7AG promoter, a 5'ALB UTR, a coding sequence for an N-terminal SV40 nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease coding sequence, and a 3'XBG UTR.

[0130] SEQ ID NO: 31 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'FGA UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'XBG UTR.

[0131] SEQ ID NO: 32 shows a DNA nucleic acid sequence comprising, from 5' to 3', a T7AG promoter, a 5'FTH1 UTR, a coding sequence for an N-terminal SV40 nuclear localization sequence, a HAO 1-2L.30S19 engineered meganuclease coding sequence, and a 3'XBG UTR.

[0132] SEQ ID NO: 33 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5' GAPDH UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3' XBG UTR.

[0133] SEQ ID NO: 34 shows a DNA nucleic acid sequence comprising, from 5' to 3', a T7AG promoter, a 5'HBA2 UTR, a coding sequence for an N-terminal SV40 nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease coding sequence, and a 3'XBG UTR.

[0134] SEQ ID NO: 35 shows a DNA nucleic acid sequence comprising, from 5' to 3', the T7AG promoter, the 5'SNRPB V1 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'XBG UTR.

[0135] SEQ ID NO: 36 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5' XBG UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3' HBA2 UTR.

[0136] SEQ ID NO: 37 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'HBB UTR.

[0137] SEQ ID NO: 38 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'SNRPB V1 UTR.

[0138] SEQ ID NO: 39 shows a DNA nucleic acid sequence comprising, from 5' to 3', the T7AG promoter, the 5'ALB UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'SNRPB V1 UTR.

[0139] SEQ ID NO: 40 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'FGA UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'SNRPB V1 UTR.

[0140] SEQ ID NO: 41 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'FTH1 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'SNRPB V1 UTR.

[0141] SEQ ID NO: 42 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5' GAPDH UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3' SNRPB V1 UTR.

[0142] SEQ ID NO: 43 shows a DNA nucleic acid sequence comprising, from 5' to 3', a T7AG promoter, a 5'HBA2 UTR, a coding sequence for an N-terminal SV40 nuclear localization sequence, a HAO 1-2L.30S19 engineered meganuclease coding sequence, and a 3'SNRPB V1 UTR.

[0143] SEQ ID NO: 44 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'SNRPB V1 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'HBA2 UTR.

[0144] SEQ ID NO: 45 shows a DNA nucleic acid sequence comprising, from 5' to 3', the T7AG promoter, the 5'SNRPB V1 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease coding sequence, and the 3'HBB UTR.

[0145] SEQ ID NO: 46 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1128 engineered meganuclease coding sequence, and the 3'WPRE UTR.

[0146] SEQ ID NO: 47 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1434 engineered meganuclease coding sequence, and the 3'WPRE UTR.

[0147] SEQ ID NO: 48 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'ALB UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1128 engineered meganuclease coding sequence, the C-terminal SV40 nuclear localization sequence, and the 3'SNRPB V1 UTR.

[0148] SEQ ID NO: 49 shows a DNA nucleic acid sequence comprising from 5' to 3' the T7AG promoter, the 5'ALB UTR, the coding sequence for the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1434 engineered meganuclease coding sequence, the C-terminal SV40 nuclear localization sequence, and the 3'SNRPB V1 UTR.

[0149] SEQ ID NO: 50 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0150] SEQ ID NO: 51 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0151] SEQ ID NO: 52 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0152] SEQ ID NO: 53 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0153] SEQ ID NO: 54 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0154] SEQ ID NO: 55 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0155] SEQ ID NO: 56 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0156] SEQ ID NO: 57 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0157] SEQ ID NO: 58 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0158] SEQ ID NO: 59 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0159] SEQ ID NO: 60 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0160] SEQ ID NO: 61 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0161] SEQ ID NO: 62 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0162] SEQ ID NO: 63 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0163] SEQ ID NO: 64 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0164] SEQ ID NO: 65 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0165] SEQ ID NO: 66 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0166] SEQ ID NO: 67 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0167] SEQ ID NO: 68 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0168] SEQ ID NO: 69 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0169] SEQ ID NO: 70 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0170] SEQ ID NO: 71 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0171] SEQ ID NO: 72 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0172] SEQ ID NO: 73 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0173] SEQ ID NO: 74 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0174] SEQ ID NO: 75 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0175] SEQ ID NO: 76 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0176] SEQ ID NO: 77 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0177] SEQ ID NO: 78 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0178] SEQ ID NO: 79 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0179] SEQ ID NO: 80 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0180] SEQ ID NO: 81 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0181] SEQ ID NO: 82 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0182] SEQ ID NO: 83 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0183] SEQ ID NO: 84 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0184] SEQ ID NO: 85 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0185] SEQ ID NO: 86 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0186] SEQ ID NO: 87 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0187] SEQ ID NO: 88 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0188] SEQ ID NO: 89 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0189] SEQ ID NO: 90 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0190] SEQ ID NO: 91 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0191] SEQ ID NO: 92 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0192] SEQ ID NO: 93 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0193] SEQ ID NO: 94 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0194] SEQ ID NO: 95 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0195] SEQ ID NO: 96 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0196] SEQ ID NO: 97 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0197] SEQ ID NO: 98 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0198] SEQ ID NO: 99 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0199] SEQ ID NO: 100 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0200] SEQ ID NO: 101 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0201] SEQ ID NO: 102 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0202] SEQ ID NO: 103 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0203] SEQ ID NO: 104 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0204] SEQ ID NO: 105 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0205] SEQ ID NO: 106 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0206] SEQ ID NO: 107 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0207] SEQ ID NO: 108 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0208] SEQ ID NO: 109 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0209] SEQ ID NO: 110 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0210] SEQ ID NO: 111 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0211] SEQ ID NO: 112 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0212] SEQ ID NO: 113 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0213] SEQ ID NO: 114 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0214] SEQ ID NO: 115 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0215] SEQ ID NO: 116 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0216] SEQ ID NO: 117 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0217] SEQ ID NO: 118 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0218] SEQ ID NO: 119 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0219] SEQ ID NO: 120 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0220] SEQ ID NO: 121 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0221] SEQ ID NO: 122 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0222] SEQ ID NO: 123 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0223] SEQ ID NO: 124 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0224] SEQ ID NO: 125 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0225] SEQ ID NO: 126 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0226] SEQ ID NO: 127 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0227] SEQ ID NO: 128 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0228] SEQ ID NO: 129 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0229] SEQ ID NO: 130 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0230] SEQ ID NO: 131 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0231] SEQ ID NO: 132 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0232] SEQ ID NO: 133 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0233] SEQ ID NO: 134 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0234] SEQ ID NO: 135 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0235] SEQ ID NO: 136 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0236] SEQ ID NO: 137 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0237] SEQ ID NO: 138 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0238] SEQ ID NO: 139 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0239] SEQ ID NO: 140 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0240] SEQ ID NO: 141 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0241] SEQ ID NO: 142 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0242] SEQ ID NO: 143 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0243] SEQ ID NO: 144 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0244] SEQ ID NO: 145 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0245] SEQ ID NO: 146 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0246] SEQ ID NO: 147 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0247] SEQ ID NO: 148 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0248] SEQ ID NO: 149 shows the DNA nucleic acid sequence of the UTR Kozak sequence.

[0249] SEQ ID NO: 150 shows the nucleic acid sequence of the ddPCR probe.

[0250] SEQ ID NO: 151 shows the nucleic acid sequence of the forward primer sequence.

[0251] SEQ ID NO: 152 shows the nucleic acid sequence of the reverse primer sequence.

[0252] SEQ ID NO: 153 shows the nucleic acid sequence of the ddPCR probe.

[0253] SEQ ID NO: 154 shows the nucleic acid sequence of the forward primer sequence.

[0254] SEQ ID NO: 155 shows the nucleic acid sequence of the reverse primer sequence.

[0255] SEQ ID NO: 156 shows the nucleic acid sequence of the ddPCR probe.

[0256] SEQ ID NO: 157 shows the nucleic acid sequence of the forward primer sequence.

[0257] SEQ ID NO: 158 shows the nucleic acid sequence of the reverse primer sequence.

[0258] SEQ ID NO: 159 shows the nucleic acid sequence of the ddPCR probe.

[0259] SEQ ID NO: 160 shows the nucleic acid sequence of the ddPCR probe.

[0260] SEQ ID NO: 161 shows the nucleic acid sequence of the ddPCR probe.

[0261] SEQ ID NO: 162 shows the nucleic acid sequence of the forward primer sequence.

[0262] SEQ ID NO: 163 shows the nucleic acid sequence of the reverse primer sequence.

[0263] SEQ ID NO: 164 shows the nucleic acid sequence of the ddPCR probe.

[0264] SEQ ID NO: 165 shows the nucleic acid sequence of the forward primer sequence.

[0265] SEQ ID NO: 166 shows the nucleic acid sequence of the reverse primer sequence.

[0266] SEQ ID NO: 167 shows the amino acid sequence of the SV40 nuclear localization sequence.

[0267] SEQ ID NO: 168 shows the DNA nucleic acid sequence encoding the SV40 nuclear localization sequence.

[0268] SEQ ID NO: 169 shows the amino acid sequence of wild-type I-Crel meganuclease.

[0269] SEQ ID NO: 170 shows the amino acid sequence of an engineered meganuclease comprising two subunits with wild-type I-Crel residues.

[0270] SEQ ID NO: 171 shows the DNA sequence of a standard control mRNA containing from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the TRC 1-2L.2307 engineered meganuclease, and the 3'WPRE UTR.

[0271] SEQ ID NO: 172 shows the DNA sequence of the mRNA, comprising from 5' to 3' the T7AG promoter, the 5'ALB UTR, the N-terminal SV40 nuclear localization sequence, the TRC 1-2L.2307 engineered meganuclease, the C-terminal SV40 nuclear localization sequence, and the 3' SNRPB V1 UTR.

[0272] SEQ ID NO: 173 shows the DNA sequence of the mRNA, containing from 5' to 3' the T7AG promoter, the 5'XBG UTR, the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease, the C-terminal SV40 nuclear localization sequence, and the 3'XBG UTR. The sequence also contains the Ssp1 linearization sequence.

[0273] SEQ ID NO: 174 shows the DNA sequence of a standard control mRNA containing, from 5' to 3', a T7AG promoter, a 5'HBA2 UTR, an N-terminal SV40 nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease, and a 3'WPRE UTR. The sequence also contains a BspQ1 linearization sequence.

[0274] SEQ ID NO: 175 shows the DNA sequence of an mRNA containing, from 5' to 3', a T7AG promoter, a 5'XBG UTR, an N-terminal SV40 nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease, a C-terminal SV40 nuclear localization sequence, and a 3'XBG UTR. The sequence also contains a BspQ1 linearization sequence.

[0275] SEQ ID NO: 176 shows the DNA sequence of an mRNA containing, from 5' to 3', a T7AG promoter, a 5'XBG UTR, an N-terminal cMyc nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease, a C-terminal cMyc nuclear localization sequence, and a 3'XBG UTR. The sequence also contains a BspQ1 linearization sequence.

[0276] SEQ ID NO: 177 shows the DNA sequence of an mRNA containing, from 5' to 3', a T7AG promoter, a 5' ALB UTR, an N-terminal SV40 nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease, a C-terminal SV40 nuclear localization sequence, and a 3' SNRPB V1 UTR. The sequence also contains a BspQ1 linearization sequence.

[0277] SEQ ID NO: 178 shows the DNA sequence of an mRNA containing, from 5' to 3', a T7AG promoter, an APT17 ribosome recruitment sequence, a 5' ALB UTR, an N-terminal SV40 nuclear localization sequence, an HAO 1-2L.30S19 engineered meganuclease, a C-terminal SV40 nuclear localization sequence, and a 3' SNRPB V1 UTR. The sequence also contains a BspQ1 linearization sequence.

[0278] SEQ ID NO: 179 shows the DNA sequence of the mRNA, comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the N-terminal cMyc nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease, the C-terminal cMyc nuclear localization sequence, and the 3'XBG UTR.

[0279] SEQ ID NO: 180 shows the DNA sequence of the mRNA, comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the N-terminal cMyc nuclear localization sequence, the HBV 11-12L.1090 engineered meganuclease, the C-terminal cMyc nuclear localization sequence, and the 3'XBG UTR.

[0280] SEQ ID NO: 181 shows the DNA sequence of a standard control mRNA containing from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the HAO 1-2L.30S19 engineered meganuclease, and the 3'WPRE UTR.

[0281] SEQ ID NO: 182 shows the DNA sequence of a standard control mRNA containing from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the HBV 11-12L.1090 engineered meganuclease, and the 3'WPRE UTR.

[0282] SEQ ID NO: 182 shows the DNA sequence of the mRNA, including from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the HBV 11-12L.1090 engineered meganuclease, and the 3'WPRE UTR.

[0283] SEQ ID NO: 183 shows the DNA sequence of the mRNA, including from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1128 engineered meganuclease, and the 3'WPRE UTR.

[0284] SEQ ID NO: 184 shows the DNA sequence of the mRNA, comprising from 5' to 3' the T7AG promoter, the 5'ALB UTR, the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1128 engineered meganuclease, the C-terminal SV40 nuclear localization sequence, and the 3' SNRPB V1 UTR.

[0285] SEQ ID NO: 185 shows the DNA sequence of the mRNA, including from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1434 engineered meganuclease, and the 3'WPRE UTR.

[0286] SEQ ID NO: 186 shows the DNA sequence of the mRNA, comprising from 5' to 3' the T7AG promoter, the 5'ALB UTR, the N-terminal SV40 nuclear localization sequence, the HAO 25-26L.1434 engineered meganuclease, the C-terminal SV40 nuclear localization sequence, and the 3'SNRPB V1 UTR.

[0287] SEQ ID NO: 187 shows the DNA sequence of the mRNA, including from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the HAO 25-26x.227 engineered meganuclease, and the 3'WPRE UTR.

[0288] SEQ ID NO: 188 shows the DNA sequence of the mRNA, including from 5' to 3' the T7AG promoter, the 5'HBA2 UTR, the N-terminal SV40 nuclear localization sequence, the TTR 15-16x.81 engineered meganuclease, and the 3'WPRE UTR.

[0289] SEQ ID NO: 189 shows the DNA sequence of the mRNA, comprising from 5' to 3' the T7AG promoter, the 5'XBG UTR, the N-terminal cMyc nuclear localization sequence, the TTR 15-16x.81 engineered meganuclease, the C-terminal cMyc nuclear localization sequence, and the 3'XBG UTR. [Brief explanation of the drawings]

[0290] [Figure 1]

[0023] Figure 1 provides a bar graph showing the percentage of indels occurring at 2.5 hours, 5 hours, and 24 hours in HEK 293 cells electroporated with 2 ng of the indicated mRNAs, as detailed in Table 1 of Example 1, encoding the HAO1-2L.30S19 engineered meganuclease. [Figure 2]3A-3D provide bar graphs showing the percentage of indels in BNL C.2 cells electroporated with either 20 ng or 200 ng of the indicated mRNA encoding the F8R17-18L.1.35 engineered meganuclease and detailed in Table 2 of Example 2. Figures 3A-3D provide bar graphs showing the percentage of indels in HEP3B cells 2, 6, and 9 days or 1, 2, 6, and 9 days after electroporation with 2 ng of the indicated mRNA encoding the HAO1-2L.30S19 engineered meganuclease and detailed in Table 3 of Example 3. [Figure 3A] Results are provided for "ON" ddPCR assays using ddPCR primers and probes set to engineered meganuclease recognition sequences at 2, 6, and 9 days after electroporation. [Figure 3B] Shown are the results of an "OFF" ddPCR assay utilizing primers and probes set away from the recognition sequence at 2, 6, and 9 days after electroporation. [Figure 3C] Results of the "ON" ddPCR assay 1, 2, 6, and 9 days after electroporation are provided. [Figure 3D] Results of "OFF" ddPCR assays 1, 2, 6, and 9 days after electroporation are provided. Figures 4A-4D provide bar graphs showing the percentage of indels occurring in HEP3B cells 2, 6, and 9 days after electroporation with 2 ng of the indicated mRNA, as detailed in Table 4 of Example 4, encoding the HAO1-2L.30S19 engineered meganuclease. [Figure 4A] Results of the "OFF" ddPCR assay on days 2, 6, and 9 are provided. [Figure 4B] Results of the "ON" ddPCR assays on days 2, 6, and 9 are provided. [Figure 4C] The data shown in Figures 4A-B are provided sorted by 5'UTR and 3'UTR combinations. [Figure 4D]Figures 4A-4B provide the data sorted by 5' UTR and 3' UTR combinations. Figures 5A-5B provide line graphs showing the percentage of indels occurring in HEP3B cells electroporated with either 0.25 ng, 0.5 ng, 1 ng, or 2 ng of the indicated mRNAs, as detailed in Table 7 of Example 5, encoding the HAO1-2L.30S19 engineered meganuclease. [Figure 5A] Results of the "OFF" ddPCR assay on days 2, 6, and 9 are provided. [Figure 5B] Results of the "ON" ddPCR assays on days 2, 6, and 9 are provided. [Figure 6]

[0023] Figure 1 provides a line graph showing the percentage of indels occurring in HepG2 cells electroporated with 0.1 ng, 0.5 ng, 2 ng, 10 ng, 50 ng, and 100 ng of the indicated mRNAs, as detailed in Table 10 of Example 6, encoding either the HAO25-26L.1434 or HAO25-26L.1128 engineered meganucleases. [Figure 7] 1 provides a graph showing engineered meganuclease protein levels in mice administered LNP formulations containing the indicated mRNA encoding the engineered meganuclease. [Figure 8] Graphs are provided showing the dose-response curves of TRC 1-2L.2307 meganuclease for knocking out cell surface CD3, as assessed by flow cytometry. The meganucleases were encoded by the optimized Max constructs disclosed herein or standard control constructs. The EC90 and EC50 values ​​for each construct are listed. [Figure 9] 1 provides a bar graph providing the percentage of indels in Hep3B cells after treatment with the indicated HAO 1-2L.30S19 meganucleases encoded by the indicated constructs. [Figure 10] 1 provides a graph showing engineered meganuclease protein levels in mice administered LNP formulations containing the indicated mRNA encoding the engineered meganuclease. [Figure 11] 1 provides a graph showing engineered meganuclease protein levels in mice administered LNP formulations containing the indicated mRNA encoding the engineered meganuclease. DETAILED DESCRIPTION OF THE INVENTION

[0291] Detailed Description of the Invention

[0292] The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. References cited herein, including issued U.S. patents, allowed applications, published foreign applications, and GenBank database sequence listings, are incorporated herein by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference.

[0293] The present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. Additionally, numerous modifications and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure without departing from the invention.

[0294] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.

[0295] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0296] I. Principles of the Invention

[0297] mRNA-based chromosome editing technology may hold the key to treating genetic diseases. However, mRNA editing platforms have room for several improvements, including extending the half-life of exogenous mRNA and, therefore, increasing the "target time" for the encoded protein to effectively edit chromosomes. Within mRNA molecules, information in the 5' and 3' untranslated regions (5' or 3' UTRs) can regulate their targeting, translation efficiency, and stability.

[0298] Provided herein are polynucleotides encoding exogenous mRNAs with regulated half-lives. The half-life can be increased or decreased to achieve optimal expression levels of the exogenous mRNA and downstream proteins. As described herein, the polynucleotides include a 5' untranslated region (UTR), a coding sequence encoding a heterologous protein, a 3' UTR, and a polyA sequence. The 5' UTR and 3' UTR can be optimized to increase the half-life of the exogenous mRNA and, consequently, increase the level of the encoded heterologous protein in eukaryotic cells. In addition, certain combinations of 5' UTRs and 3' UTRs can reduce the persistence of exogenous mRNA molecules. As experimentally described and demonstrated herein, certain combinations of UTRs result in higher levels of expression than other combinations. Thus, the combination of 5' UTRs and 3' UTRs allows for the tunability of mRNA persistence, and therefore downstream heterologous protein expression. In some embodiments, the heterologous protein is an engineered nuclease, e.g., an engineered meganuclease. In some embodiments, the genome editing efficiency of the engineered nuclease is advantageously increased compared to the control mRNA construct. In other embodiments, the genome editing efficiency is advantageously decreased compared to the control mRNA construct. Also provided herein are pharmaceutical compositions comprising the polynucleotide, methods for expressing heterologous proteins in eukaryotic cells using the polynucleotide, and methods for treating diseases in subjects using the pharmaceutical compositions.

[0299] II. Definition

[0300] As used herein, "a," "an," or "the" can mean one or more. For example, "a" cell can mean a single cell or many cells.

[0301] As used herein, unless otherwise expressly stated, the word "or" is used in the inclusive sense of "and / or" and not in the exclusive sense of "either / or."

[0302] As used herein, all polynucleotide sequences written using the International Union of Pure and Applied Chemistry (IUPAC, Biochemistry (1970) Vol. 9:4022-4027) standard notation for nucleic acids (adenine (A), thymine (T), guanine (G), and cytosine (C)) are equivalent to the corresponding RNA polynucleotide sequences. Thus, "T" (thymine) in all sequences is equivalent to "U" (uracil). For example, the sequence AATAAA in a DNA coding strand is also considered to represent the corresponding mRNA sequence AAUAAA.

[0303] As used herein, the use of the terms "polynucleotide," "DNA," or "nucleic acid" is not intended to limit the present invention to polynucleotides that contain DNA. Those skilled in the art will recognize that polynucleotides can contain ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present invention also encompass all forms of sequences, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0304] As used herein, the term "5' untranslated region" or "5' UTR" refers to the region of a messenger RNA (mRNA) immediately upstream of the start codon. This region is important for regulating the translation of transcripts by different mechanisms in viruses, prokaryotes, and eukaryotes. Although called untranslated, the 5' UTR or a portion thereof can, in some cases, be translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA. However, in many organisms, the 5' UTR is not fully translated, but instead forms complex secondary structures that can regulate translation.

[0305] The average length of a 5' UTR ranges from approximately 30 to approximately 220 nucleotides across species. In vertebrates, 5' UTRs tend to be longer in transcripts encoding transcription factors, protooncogenes, growth factors, and their receptors, as well as proteins that are poorly translated under normal conditions. High GC content is also a conserved feature of 5' UTRs, exceeding 60% in warm-blooded vertebrates. In relation to hairpin structures, GC content can affect protein translation efficiency independently of hairpin thermostability and hairpin position. UTRs of eukaryotic mRNAs also exhibit a variety of repetitive sequences, including short and long interspersed sequences (SINEs and LINEs, respectively), simple sequence repeats (SSRs), minisatellites, and macrosatellites. Translation initiation in eukaryotes requires the recruitment of ribosomal subunits at either the 5' m7G cap structure. Genes exhibiting variations in the 5' UTR of transcripts are relatively common. While 10-18% of genes express alternative 5'UTRs by using multiple promoters, alternative splicing within UTRs is estimated to affect 13% of genes in the mammalian transcriptome. These mutations in 5'UTRs can function as important switches for regulating gene expression. 5'UTRs can form secondary structures, namely hairpin loops, that affect the regulation of translation.

[0306] In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing the heterologous protein start codon. In some embodiments, the 5' UTR does not form a stable secondary sequence structure containing the heterologous protein start codon with a change in free energy (ΔG) of less than about -10 kcal / mol to about -80 kcal / mol. In certain embodiments, the change in free energy is less than about -5 kcal / mol, -10 kcal / mol, -20 kcal / mol, -30 kcal / mol, -40 kcal / mol, -50 kcal / mol, -60 kcal / mol, -70 kcal / mol, -80 kcal / mol, -90 kcal / mol, or less than about -100 kcal / mol. In some embodiments, the 5' UTR comprises an internal ribosome entry site (IRES).

[0307] In some embodiments, the 5'UTR is the 5'UTR of the ALB gene (SEQ ID NO: 1), or the FGA gene (SEQ ID NO: 2), or the 5'UTR of the FTH1 gene (SEQ ID NO: 3), or the 5'UTR of the GAPDH gene (SEQ ID NO: 4), or the 5'UTR of the HBA2 gene (SEQ ID NO: 5), or the 5'UTR of SNRPB variant 1 (SEQ ID NO: 6), or the 5'UTR of the XBG gene (SEQ ID NO: 7). In various embodiments, the 5'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7. In various embodiments, the 5'UTR is any one of SEQ ID NOs: 1-7. In various embodiments, the 5' UTR comprises a UTR Kozak sequence. In some embodiments, the UTR Kozak sequence is any one of SEQ ID NOs: 50-149. In particular embodiments, the UTR Kozak sequence comprises SEQ ID NO: 114. In some embodiments, the 5' UTR comprises a eukaryotic initiation factor (eIF) recruitment sequence.

[0308] As used herein, the term "3' untranslated region" or "3'UTR" refers to the portion of messenger RNA (mRNA) immediately following the translation termination codon. On average, the length of 3'UTR in humans is about 800 nucleotides. The length of 3'UTR is important because longer 3'UTRs are associated with lower levels of gene expression. One possible explanation for this phenomenon is that longer regions are more likely to have more miRNA binding sites that have the ability to inhibit translation.

[0309] 3'UTRs often contain regulatory regions that influence gene expression after transcription. Regulatory regions within 3'UTRs can affect mRNA polyadenylation, translation efficiency, localization, and stability. 3'UTRs can contain binding sites for both regulatory proteins and microRNAs (miRNAs). By binding to specific sites within the 3'UTR, miRNAs can reduce gene expression of various mRNAs by either inhibiting translation or directly causing transcript degradation. 3'UTRs can also contain silencer regions that bind repressor proteins and inhibit mRNA expression. Many 3'UTRs also contain AU-rich regions (AREs). Proteins bind to AREs to locally affect transcript stability or decay rate or to affect translation initiation. Additionally, 3'UTRs can contain the sequence AAUAAA, which directs the addition of several hundred adenine residues, called a poly(A) tail, to the end of mRNA transcripts. Poly(A)-binding proteins (PABPs) bind to this tail and contribute to the regulation of mRNA translation, stability, and export. For example, PABPs bound to the poly(A) tail interact with proteins associated with the 5' end of the transcript, causing mRNA circularization, which promotes translation. The 3' UTR may also contain sequences that attract proteins to associate the mRNA with the cytoskeleton, transport it to or from the nucleus, or perform other types of localization. In addition to the sequence within the 3' UTR, the physical properties of the region, including its length and secondary structure, contribute to the regulation of translation. These diverse mechanisms of gene regulation ensure that the correct gene is expressed in the correct cell at the correct time.

[0310] In various embodiments, the 3'UTR is the 3'UTR of the HBA2 gene (SEQ ID NO:8), or the 3'UTR of the HBB gene (SEQ ID NO:9), or the 3'UTR of SNRPB variant 1 (SEQ ID NO:10), or the 3'UTR of SNRPB variant 2 (SEQ ID NO:11), or the 3'UTR of the gene XBG (SEQ ID NO:12), or the 3'UTR of the gene WPRE (SEQ ID NO:13). In some embodiments, the 3'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to any one of SEQ ID NOs:8, 9, 10, 11, 12, or 13.

[0311] As used herein, the term "Kozak sequence" refers to a nucleic acid motif that functions as a protein translation initiation site in most eukaryotic mRNA transcripts. Vertebrate Kozak sequences have a consensus sequence of "gcc A / G ccATGG" (SEQ ID NO: 190), with capital letter positions being more conserved than lowercase letters, and ATG being the start codon. Thus, the Kozak sequence spans the 5' UTR and the coding sequence, and the portion within the 5' UTR is the UTR Kozak sequence. For example, the UTR Kozak sequence is the portion of the Kozak sequence from base pairs 1 through 6. In various embodiments, the first nucleotide of the Kozak sequence is A or G. In various embodiments, the second nucleotide of the Kozak sequence is C or T. In various embodiments, the third nucleotide of the Kozak sequence is A or C. In various embodiments, the fourth nucleotide of the Kozak sequence is A or G. In various embodiments, the fifth nucleotide of the Kozak sequence is A or C. In various embodiments, the sixth nucleotide of the Kozak sequence is A, C, or G. In certain embodiments, the Kozak sequence comprises the sequence GCCACC, which is part of the 5'UTR. In various embodiments, the seventh through tenth nucleotides of the Kozak sequence are ATGG. In certain embodiments, the Kozak sequence can comprise a portion of the NLS of a polynucleotide. For example, the Kozak sequence can comprise the sequence ATGGC, which is part of the SV40 NLS. In various embodiments, the UTR Kozak sequence comprises any one of SEQ ID NOs: 50-149.

[0312] As used herein, the term "GC content" refers to the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). This measure indicates the proportion of G and C bases out of the four implied bases, including adenine and thymine in DNA and adenine and uracil in RNA. DNA with a low GC content is less stable than DNA with a high GC content, but hydrogen bonding itself does not have a particularly large effect on the molecule's stability; instead, base-stacking molecular interactions are the primary cause.

[0313] As used herein, the terms "adenine or thymine content" or "AT content" refer to the percentage of nitrogenous bases in DNA that are either adenine (A) or thymine (T), or the percentage of nitrogenous bases in an RNA molecule that are either adenine (A) or uracil (U). This measure indicates the proportion of A and T bases out of the four total implied bases in DNA, or the proportion of A and U bases out of the four total implied bases in RNA.

[0314] As used herein, the term "5' cap" refers to a specially altered nucleotide on the 5' end of some primary transcripts, such as precursor messenger RNAs. This process, known as mRNA capping, is highly regulated and essential for the creation of stable, mature messenger RNAs that can undergo translation during protein synthesis. Mitochondrial and chloroplast mRNAs are not capped. In eukaryotes, the 5' cap found at the 5' end of mRNA molecules consists of a guanosine nucleotide attached to the mRNA via an unusual 5'-to-5' triphosphate linkage. This guanosine is methylated at the 7th position immediately after in vivo capping by a methyltransferase. This is called the 7-methylguanylate cap, abbreviated as m7G. In multicellular eukaryotes and some viruses, additional modifications occur, including methylation of the 2' hydroxyl groups of the first two ribose sugars at the 5' end of mRNAs. Cap-1 has a methylated 2' hydroxy group on the first ribose sugar, while Cap-2 has methylated 2' hydroxy groups on the first two ribose sugars, as shown on the right. The 5' cap chemically resembles the 3' end of an RNA molecule (the 5' carbon of the cap ribose is bound, and the 3' is unbound). This confers significant resistance to 5' exonucleases.

[0315] As used herein, the term "indel" is a molecular biology term for the insertion or deletion of a base in the genome of an organism. In the coding region of a genome, unless the length of the indel is a multiple of three, a frameshift mutation occurs. Indels can be contrasted with point mutations. Indels cause nucleotides to be inserted and deleted from a sequence, while point mutations are a form of substitution that replaces one nucleotide without changing the total number in DNA. Indels can also be contrasted with tandem base mutations (TBMs), which can arise from fundamentally different mechanisms. Indels, either insertions or deletions, can be used as genetic markers in natural populations, especially in phylogenetic studies (Vali et al., BMC Genet., 2008;9:8; Erixon et al., PLoS One, 2008;3(1):e1386). Indel percentage can be measured using various methods, for example, using ddPCR. Indel percentage can be used to evaluate the genome editing efficiency of engineered nucleases. For example, the indel percentage can be used to assess the genome editing efficiency of any engineered nuclease used in the present invention, including but not limited to, engineered meganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPR system nucleases, and megaTALs.

[0316] As used herein, the terms "heterologous" or "exogenous" with respect to a nucleotide sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that is derived from a foreign species, or, if derived from the same species, that has been substantially modified in composition and / or genomic locus from its native form by deliberate human intervention.

[0317] As used herein, the term "endogenous" with respect to a nucleotide sequence or protein is intended to mean a sequence or protein that is naturally contained within or expressed by a cell.

[0318] As used herein, the term "modification" with respect to a polynucleotide refers to any insertion, deletion, or substitution of one or more base pairs in the polynucleotide. In some embodiments, the modification is applied to the coding sequence of a heterologous protein without changing the amino acid sequence of the heterologous protein. In some embodiments, the heterologous protein is an engineered nuclease. In some embodiments, the modification of the coding sequence of a heterologous protein comprises changing a first three-base codon containing thymidine or uridine to a second three-base codon with less thymidine or uridine without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification of the coding sequence of a heterologous protein comprises changing a first three-base codon containing thymidine or uridine to a second three-base codon without thymidine or uridine without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification decreases the thymidine or uridine content of the coding sequence. In some embodiments, the modification increases the guanine or cytosine content of the coding sequence. In some embodiments, the coding sequence has a 10% to 90%, or 20% to 80%, or 30% to 70%, or 40% to 60%, or 45% to 55% reduced thymidine or uridine content compared to a coding sequence that has not been modified to reduce thymidine or uridine content. In some embodiments, the coding sequence has a 40% reduced thymidine or uridine content compared to a coding sequence that has not been modified to reduce thymidine or uridine content. In various embodiments, the modification does not alter the protein levels of the heterologous protein. In some embodiments, the modification results in enhanced expression of the heterologous protein. In some embodiments, the modification can enhance expression of the heterologous protein by at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 200%, 500%, 1000%, or more compared to without the modification.

[0319] As used herein, the terms "AU-rich region," "adenylate-uridylate-rich region," or "ARE" refer to nucleic acid sequences found in the 3' untranslated region (UTR) of many mRNAs encoding proto-oncogenes, nuclear transcription factors, and cytokines. AREs are one of the most common determinants of RNA stability in mammalian cells. AREs are defined as regions with a high frequency of adenine and uridine bases in mRNA. AREs typically target mRNAs for rapid degradation. AREs have been divided into three classes with distinct sequences. The best-characterized AREs have a core sequence of AUUUA within a U-rich sequence (e.g., WWWU(AUUUA)UUUW, where W is A or U). This is within a 50-150 base sequence, and repeats of the core AUUUA element are often required for function. Class I AREs, like those in the c-fos gene, have AUUUA motifs dispersed within or near U-rich regions. Class II AREs, like those in the GM-CSF gene, have overlapping AUUUA motifs within or near U-rich regions. Class III elements, like those in the c-jun gene, are a much less well-defined class that contain U-rich regions but no AUUUA repeats.

[0320] As used herein, the term "open reading frame" refers to a portion of a DNA molecule that does not contain a stop codon when translated into amino acids. The genetic code reads the DNA sequence of groups of three base pairs, which means that a double-stranded DNA molecule can be read in one of six possible reading frames (three forward and three reverse). A long open reading frame is likely to be part of a gene.

[0321] As used herein, the term "eukaryotic initiation factor (eIF) recruitment sequence" or "eIF recruitment sequence" refers to a sequence within a 5'UTR to which eIF binds. In some embodiments, the eIF recruitment sequence comprises an eIF4G recruitment sequence. In some embodiments, the eIF4G recruitment sequence comprises APT17. In some embodiments, the APT17 sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 14.

[0322] As used herein, the term "nuclear localization sequence" or "NLS" refers to a generally short peptide that acts as a signal fragment mediating the transport of proteins from the cytoplasm to the nucleus. Classical NLSs encompass two categories: monopartite (MP) and bipartite NLSs. Monopartite NLSs have a single cluster of 4-8 basic amino acids, which generally contain four or more positively charged residues, i.e., arginine (R) or lysine (K). The characteristic motif of MP NLSs is usually defined as K(K / R)X(K / R), where X can be any residue. For example, the NLS of the SV40 large T antigen is 126PKKKRKV132 (SEQ ID NO: 15), which has five consecutive positively charged amino acids (KKKRK) (SEQ ID NO: 191). Bipartite NLSs are characterized by two clusters of 2-3 positively charged amino acids separated by a 9-12 amino acid linker region containing several proline (P) residues. The consensus sequence can be represented as R / K(X)10-12KRXK. In particular, in bipartite NLSs, the upstream and downstream clusters of amino acids are interdependent and essential, jointly determining the subcellular localization of the protein. Nonclassical nuclear localization sequences do not resemble canonical signals, nor are they rich in arginine or lysine residues. Among nonclassical nuclear localization sequences, the "proline-tyrosine" category has been studied most extensively. PY-NLSs are characterized by an irregular structure of 20-30 amino acids, consisting of an N-terminal hydrophobic or basic motif and a C-terminal R / K / H(X)2-5PY motif (X2-5 is any sequence of 2-5 residues). Two subclasses, hPY-NLSs and bPY-NLSs, have been defined according to their N-terminal motifs. hPY-NLSs contain a φG / A / Sφφ motif (φ is a hydrophobic residue), whereas bPY-NLSs are rich in basic residues. In summary, the PY-NLS consensus corresponds to [basic / hydrophobic]-Xn-[R / H / K]-(X)2-5-PY, where X can be any residue.Human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is known as hPY-NLS because of its sequence 263FGNYNNQSSNFGPMKGGNFGGRSSGPY289 (SEQ ID NO: 192), which contains a hydrophobic region (273FGPM276) (SEQ ID NO: 193) necessary for its nuclear localization.

[0323] In some embodiments, the NLS comprises an SV40 NLS (SEQ ID NO: 15 or 19), NLS5 (SEQ ID NO: 16 or 20), a CMYC NLS (SEQ ID NO: 17), or an SV40H2 NLS (SEQ ID NO: 18). In some embodiments, the NLS comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 15-20. In some embodiments, the NLS comprises the amino acid sequence of any one of SEQ ID NOs: 15-20.

[0324] As used herein, the term "wild-type" refers to the most common naturally occurring allele (i.e., polynucleotide sequence) among a population of alleles of the same type of gene, and a polypeptide encoded by a wild-type allele retains its original function. The term "wild-type" also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides that contain one or more mutations and / or substitutions compared to the wild-type sequence. A wild-type allele or polypeptide can confer a normal phenotype in an organism, while a mutant or variant allele or polypeptide can, in some cases, confer an altered phenotype. A wild-type nuclease is distinguishable from a recombinant nuclease or a non-naturally occurring nuclease. The term "wild-type" can also refer to a cell, organism, and / or subject that has a wild-type allele of a particular gene, or a cell, organism, and / or subject used for comparison purposes.

[0325] As used herein, the terms "percent identity," "sequence identity," "percentage of similarity," "sequence similarity," and the like, for both amino acid and nucleic acid sequences, refer to a measure of the degree of similarity between two sequences based on the alignment of sequences that maximizes the similarity between aligned amino acid residues or nucleotides, and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) and are described, for example, in Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol. 266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402; Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As used herein, the percent similarity of two amino acid sequences is a score based on the following parameters for the BLASTp algorithm: word size=3, gap opening penalty=-11, gap extension penalty=-1, and score matrix=BLOSUM62. As used herein, the percent similarity of two nucleic acid sequences is a score based on the following parameters for the BLASTn algorithm: word size=11, gap opening penalty=-5, gap extension penalty=-2, match reward=1, and mismatch penalty=-3.

[0326] As used herein, the terms "recombinant DNA construct," "recombinant construct," "expression cassette," "expression construct," "chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably herein and refer to single- or double-stranded polynucleotides. Recombinant constructs include artificial combinations of nucleic acid fragments, including, but not limited to, regulatory and coding sequences not found together in nature. For example, recombinant DNA constructs can contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found in nature. Such constructs can be used alone or in conjunction with a vector. In some embodiments, the recombinant DNA construct is a plasmid.

[0327] As used herein, the terms "treatment," "treating," or "treating a subject" refer to the administration of a therapeutically effective amount of a pharmaceutical composition disclosed herein comprising a polynucleotide described herein, and the heterologous protein is a therapeutic protein. For example, the subject may have a disease, such as a genetic disease, and treatment can refer to genetic therapy for treating the disease. Desirable effects of treatment include, but are not limited to, correcting a disease-associated mutation in the subject, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improving prognosis. In some embodiments, treatment involves administering to a subject in need thereof nanoparticles comprising a pharmaceutical composition described herein. In various embodiments, the heterologous protein is an engineered nuclease. In various embodiments, the engineered nuclease increases protein levels in eukaryotic cells. In various embodiments, the engineered nuclease results in indels in eukaryotic cells.

[0328] As used herein, the term "control polynucleotide" refers to a polynucleotide that encodes a heterologous protein described herein, but does not include a 5'UTR, or a 3'UTR, or both, or does not include a 5'UTR, or a 3'UTR, or both, as described herein. In some embodiments, the control polynucleotide is an mRNA. In some embodiments, the control polynucleotide is a recombinant DNA construct. In some embodiments, the control polynucleotide is introduced into a eukaryotic cell by a lipid nanoparticle. In some embodiments, the control polynucleotide is introduced into a eukaryotic cell by a recombinant virus.

[0329] In various embodiments, the control polynucleotide does not include the 5' UTR of the ALB gene, or the FGA gene, or the FTH1 gene, or the GAPDH gene, or the HBA2 gene, or the SNRPB V1 gene, or the SNRPB1 gene, or the XBG gene. In various embodiments, the control polynucleotide does not include a 5' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 1-7. In various embodiments, the control polynucleotide does not include a UTR Kozak sequence. In some embodiments, the control polynucleotide does not contain a UTR Kozak sequence that is any one of SEQ ID NOS: 50-149. In various embodiments, the control polynucleotide does not contain the 3' UTR of the HBA2 gene, or the 3' UTR of the SNRPB V1 gene, or the 3' UTR of the SNRPB V2 gene, or the 3' UTR of the WPRE gene, or the 3' UTR of the XBG gene. In various embodiments, the control polynucleotide does not contain a 3' UTR that has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOS: 8-13. In various embodiments, the control polynucleotide does not include a 3'UTR that is any one of SEQ ID NOs: 8-13.

[0330] In some embodiments, the control polynucleotide does not contain an NLS. In some embodiments, the control polynucleotide does not contain an NLS comprising an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 15, 16, 17, or 18. In some embodiments, the NLS comprises the amino acid sequence of any one of SEQ ID NOs: 15-18. In some embodiments, the control polynucleotide does not contain an NLS comprising the amino acid sequence of any one of SEQ ID NOs: 15-18.

[0331] In various embodiments, the control polynucleotide does not contain pseudouridine or 2-thiouridine. In various embodiments, the control polynucleotide is unmethylated. In various embodiments, the control polynucleotide does not contain N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0332] In some embodiments, the control polynucleotide comprises the 5' UTR of the HBA2 gene (i.e., SEQ ID NO: 5) and the 3' UTR of the WPRE gene (i.e., SEQ ID NO: 13). In some embodiments, the control polynucleotide comprises an SV40 NLS (i.e., SEQ ID NO: 15). In some embodiments, the control polynucleotide comprises an N-terminal SV40 NLS (i.e., SEQ ID NO: 15). In some embodiments, the control polynucleotide comprises a C-terminal SV40 NLS (i.e., SEQ ID NO: 15). In some embodiments, the control polynucleotide comprises an N-terminal SV40 NLS (i.e., SEQ ID NO: 15), the 5' UTR of the HBA2 gene (i.e., SEQ ID NO: 5), and the 3' UTR of the WPRE gene (i.e., SEQ ID NO: 13).

[0333] As used herein, the term "control cell" refers to a cell containing a control polynucleotide. The control cell can provide a reference point for measuring fold changes in heterologous protein levels or mRNA persistence. In some embodiments, the protein level of the heterologous protein is increased by about 2-10 fold in the eukaryotic cell compared to the control eukaryotic cell. In various embodiments, mRNA persistence is improved by about 2-10 fold in the eukaryotic cell compared to the control eukaryotic cell. In some embodiments, the control cell is a mammalian cell. In some embodiments, the control cell is a human cell. In some embodiments, the control cell is part of a tissue. In some embodiments, the control cell is within a mammal. In some embodiments, the control cell is within a human.

[0334] As used herein, the term "effective amount" or "therapeutically effective amount" of a pharmaceutical composition refers to an amount sufficient to produce a beneficial or desired result, e.g., in curing, alleviating, mitigating, or ameliorating one or more symptoms, clinical outcomes, of a disorder, upon single or multiple administration to a subject's cells; thus, "effective amount" depends on the context in which it is applied. For example, in the context of administering a drug to treat a genetic disease, an effective amount of a pharmaceutical composition is an amount sufficient to achieve treatment of the genetic disease, as defined herein, compared to, e.g., the response obtained without administration of the pharmaceutical composition.

[0335] As used herein, the term "vector" or "recombinant DNA vector" refers to a construct containing a replication system and sequences capable of transcribing and translating a polypeptide-encoding sequence in a given host cell. When a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include, but are not limited to, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering genes to target cells. Those skilled in the art are well aware of the genetic elements that must be present on a vector to successfully transform, select, and propagate host cells containing any of the isolated nucleotide or nucleic acid sequences of the present invention. In some embodiments, "vector" also refers to a virus (i.e., a viral vector). Viruses include, but are not limited to, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs). In some embodiments, a vector may refer to a plasmid.

[0336] III. Engineered Nucleases

[0337] As described herein, heterologous protein can be engineered nuclease.Any engineered nuclease can be used in the methods and compositions disclosed herein, including engineered meganuclease, zinc finger nuclease, TALEN, compact TALEN, CRISPR system nuclease or megaTAL.

[0338] For example, zinc finger nucleases (ZFNs) can be engineered to recognize and cleave specific sites within the genome. ZFNs are chimeric proteins containing a zinc finger DNA binding domain fused to a nuclease domain derived from an endonuclease or exonuclease (e.g., a type II restriction endonuclease such as FokI restriction enzyme). The zinc finger domain can be a natural sequence or can be redesigned by rational or experimental means to produce a protein that binds to a specific DNA sequence approximately 18 base pairs in length. By fusing this engineered protein domain to a nuclease domain, it is possible to target DNA cleavage with genome-level specificity. ZFNs have been widely used to target gene addition, removal, and replacement in a wide range of eukaryotic organisms (reviewed in S. Durai et al., Nucleic Acids Res., 2005, 33, 5978).

[0339] Similarly, TAL-effector nuclease (TALEN) can be generated to cut specific sites in genomic DNA.Like ZFN, TALEN comprises engineered site-specific DNA binding domain fused to endonuclease or exonuclease (for example, type II restriction endonuclease such as FokI restriction enzyme) (reviewed in Mak, et al., Curr Opin Struct Biol., 2013,23:93-9).However, in this case, DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair.

[0340] Compact TALEN is an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley, et al., Nat Commun., 2013, 4:1762). Compact TALENs comprise engineered site-specific TAL-effector DNA binding domains fused to the nuclease domains derived from I-Tevl homing endonucleases or any of the endonucleases listed in Table 2 of US Patent Application No. 20130117869. Compact TALENs do not require dimerization for DNA processing activity, so compact TALENs function as monomers.

[0341] Engineered endonucleases based on the CRISPR / Cas system are also known in the art (Ran, et al., Nat Protoc., 2013, 8:2281-2308; Mali et al., Nat Methods., 2013, 10:957-63). The CRISPR system includes two components: (1) a CRISPR nuclease, and (2) a short "guide RNA" that includes a targeting sequence of about 20 nucleotides, which guides the nuclease to the desired location in the genome. The CRISPR system can also include tracrRNA. By expressing multiple guide RNAs, each with a different targeting sequence, in the same cell, it is possible to simultaneously target DNA cleavage to multiple sites in the genome.

[0342] The method of the present disclosure can be used to use engineered meganucleases that bind to double-stranded DNA with a recognition sequence of more than 12 base pairs. Meganucleases can be endonucleases derived from I-Crel, and can refer to engineered variants of I-Crel that are modified relative to natural I-Crel, for example, in terms of DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties. Methods for producing such modified variants of I-Crel are known in the art (e.g., International Publication No. 2007 / 047859, the entirety of which is incorporated by reference). Meganucleases used herein bind to double-stranded DNA as heterodimers. Meganucleases can also be "single-chain meganucleases," in which a pair of DNA binding domains are linked to a single polypeptide using a peptide linker.

[0343] The nuclease, termed megaTAL, is a single-stranded endonuclease containing a transcription activator-like effector (TALE) DNA-binding domain with an engineered sequence-specific homing endonuclease.

[0344] In certain embodiments, the nuclease used to implement the present invention is a single-chain meganuclease. Single-chain meganucleases comprise an N-terminal subunit and a C-terminal subunit connected by a linker peptide. Each of the two domains recognizes half of a recognition sequence (i.e., a recognition half-site), and the DNA cleavage site is located in the center of the recognition sequence near the interface between the two subunits. DNA strand cleavage is offset by four base pairs, so that DNA cleavage by the meganuclease generates a pair of four-base pair, 3' single-stranded overhangs. For example, nuclease-mediated insertion using engineered single-chain meganucleases is disclosed in International Publication Nos. 2017 / 062439 and 2017 / 062451.

[0345] IV. mRNA-based chromosome editing platform

[0346] Provided herein is a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein, the nucleic acid sequence comprising a 5' UTR, a coding sequence encoding the heterologous protein, a 3' UTR, and a polyA sequence. In various embodiments, the polynucleotide does not comprise an upstream uATG sequence or an upstream open reading frame sequence.

[0347] In some embodiments, the 5'UTR is the 5'UTR of the ALB gene (SEQ ID NO: 1), or the FGA gene (SEQ ID NO: 2), or the 5'UTR of the FTH1 gene (SEQ ID NO: 3), or the 5'UTR of the GAPDH gene (SEQ ID NO: 4), or the 5'UTR of the HBA2 gene (SEQ ID NO: 5), or the 5'UTR of SNRPB variant 1 (SEQ ID NO: 6), or the 5'UTR of the XBG gene (SEQ ID NO: 7). In various embodiments, the 5'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to any one of SEQ ID NOs: 1-7. In various embodiments, the 5' UTR is any one of SEQ ID NOs: 1-7. In various embodiments, the 5' UTR comprises a UTR Kozak sequence. In some embodiments, the UTR Kozak sequence has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 50-149. In some specific embodiments, the UTR Kozak sequence comprises any one of SEQ ID NOs: 50-149. In certain embodiments, the UTR Kozak sequence comprises SEQ ID NO: 114.

[0348] In certain embodiments, the 3'UTR is the 3'UTR of the HBA2 gene (SEQ ID NO:8), or the 3'UTR of the HBB gene (SEQ ID NO:9), or the 3'UTR of SNRPB variant 1 (SEQ ID NO:10), or the 3'UTR of SNRPB variant 2 (SEQ ID NO:11), or the 3'UTR of the gene XBG (SEQ ID NO:12), or the 3'UTR of the gene WPRE (SEQ ID NO:13). In various embodiments, the 3'UTR has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to any one of SEQ ID NOs:8-13. In various embodiments, the 3'UTR is any one of SEQ ID NOs: 8-13.

[0349] In various embodiments, the polynucleotide comprises any combination of 5'UTR and 3'UTR. For example, in some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the ALB gene (SEQ ID NO: 1). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the ALB gene (SEQ ID NO: 1) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8).

[0350] In some embodiments, the polynucleotide has a 5' UTR that has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5' UTR of the ALB gene (SEQ ID NO: 1). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the ALB gene (SEQ ID NO: 1) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9), wherein the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0351] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the ALB gene (SEQ ID NO: 1). In certain embodiments, the polynucleotide comprises a 3' UTR having at least at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3' UTR of SNRPB variant 1 (SEQ ID NO: 10). In certain embodiments, the polynucleotide comprises a 5' UTR comprising the 5' UTR of the ALB gene (SEQ ID NO: 1) and a 3' UTR comprising the 3' UTR of SNRPB variant 1 (SEQ ID NO: 10).

[0352] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the ALB gene (SEQ ID NO: 1). and the polynucleotide comprises a 3' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3' UTR of SNRPB variant 2 (SEQ ID NO: 11). In certain embodiments, the polynucleotide comprises a 5' UTR comprising the 5' UTR of the ALB gene (SEQ ID NO: 1) and a 3' UTR comprising the 3' UTR of SNRPB variant 2 (SEQ ID NO: 11).

[0353] In some embodiments, the polynucleotide has a 5' UTR that has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5' UTR of the ALB gene (SEQ ID NO: 1). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the ALB gene (SEQ ID NO: 1) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12), wherein the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene XBG (SEQ ID NO: 12).

[0354] In some embodiments, the polynucleotide has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the ALB gene (SEQ ID NO: 1). R, wherein the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of gene WPRE (SEQ ID NO: 13). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the ALB gene (SEQ ID NO: 1) and a 3'UTR comprising the 3'UTR of gene WPRE (SEQ ID NO: 13).

[0355] In some embodiments, the polynucleotide has a 5'UTR that has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FGA gene (SEQ ID NO: 2). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FGA gene (SEQ ID NO:2) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO:8), wherein the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO:8).

[0356] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FGA gene (SEQ ID NO:2), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO:9). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FGA gene (SEQ ID NO: 2) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0357] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FGA gene (SEQ ID NO:2), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRPB variant 1 (SEQ ID NO:10). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FGA gene (SEQ ID NO: 2) and a 3'UTR comprising the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10).

[0358] In some embodiments, the polynucleotide comprises a 5' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5' UTR of the FGA gene (SEQ ID NO: 2), and the polynucleotide comprises a 3' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3' UTR of SNRPB variant 2 (SEQ ID NO: 11). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FGA gene (SEQ ID NO: 2) and a 3'UTR comprising the 3'UTR of SNRPB variant 2 (SEQ ID NO: 11).

[0359] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FGA gene (SEQ ID NO: 2), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene XBG (SEQ ID NO: 12). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FGA gene (SEQ ID NO: 2) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12).

[0360] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FGA gene (SEQ ID NO: 2), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FGA gene (SEQ ID NO: 2) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0361] In some embodiments, the polynucleotide is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,

[0362] The polynucleotide comprises a 5'UTR having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity, and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FTH1 gene (SEQ ID NO: 3) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8).

[0363] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FTH1 gene (SEQ ID NO: 3), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO: 9). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FTH1 gene (SEQ ID NO: 3) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0364] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FTH1 gene (SEQ ID NO: 3), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FTH1 gene (SEQ ID NO: 3) and a 3'UTR comprising the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10).

[0365] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FTH1 gene (SEQ ID NO: 3), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRPB variant 2 (SEQ ID NO: 11). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FTH1 gene (SEQ ID NO: 3) and a 3'UTR comprising the 3'UTR of SNRPB variant 2 (SEQ ID NO: 11).

[0366] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FTH1 gene (SEQ ID NO: 3), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene XBG (SEQ ID NO: 12). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FTH1 gene (SEQ ID NO: 3) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12).

[0367] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the FTH1 gene (SEQ ID NO: 3), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the FTH1 gene (SEQ ID NO: 3) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0368] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the GAPDH gene (SEQ ID NO: 4), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the GAPDH gene (SEQ ID NO: 4) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8).

[0369] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the GAPDH gene (SEQ ID NO: 4), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO: 9). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the GAPDH gene (SEQ ID NO: 4) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0370] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the GAPDH gene (SEQ ID NO: 4), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the GAPDH gene (SEQ ID NO: 4) and a 3'UTR comprising the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10).

[0371] In some embodiments, the polynucleotide comprises a 5' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5' UTR of the GAPDH gene (SEQ ID NO: 4), and the polynucleotide comprises a 3' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3' UTR of SNRPB variant 2 (SEQ ID NO: 11). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the GAPDH gene (SEQ ID NO: 4) and a 3'UTR comprising the 3'UTR of SNRPB variant 2 (SEQ ID NO: 11).

[0372] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the GAPDH gene (SEQ ID NO: 4), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene XBG (SEQ ID NO: 12). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the GAPDH gene (SEQ ID NO: 4) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12).

[0373] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the GAPDH gene (SEQ ID NO: 4), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the GAPDH gene (SEQ ID NO: 4) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0374] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In some embodiments, a polynucleotide comprising a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8) can be used to reduce protein expression and / or activity.In certain embodiments, a polynucleotide comprising a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8) can be used to reduce protein expression and / or activity.

[0375] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO: 9). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0376] In some embodiments, the polynucleotide comprises a 5' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5' UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3' UTR of SNRPB variant 1 (SEQ ID NO: 10). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10).

[0377] In some embodiments, the polynucleotide comprises a 5' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5' UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3' UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3' UTR of SNRPB variant 2 (SEQ ID NO: 11). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of SNRPB variant 2 (SEQ ID NO: 11). In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene XBG (SEQ ID NO: 12). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12). In some embodiments, a polynucleotide comprising a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the XBG gene (SEQ ID NO: 12) can be used to reduce protein expression and / or activity.In certain embodiments, a polynucleotide comprising a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the XBG gene (SEQ ID NO: 12) can be used to reduce protein expression and / or activity.

[0378] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13). In some embodiments, the control polynucleotides described herein comprise a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the HBA2 gene (SEQ ID NO: 5), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13).In certain embodiments, the control polynucleotides described herein comprise the 5'UTR of the HBA2 gene (SEQ ID NO: 5) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0379] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6), and the polynucleotide comprises a 3'UTR having at least 60%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO: 8). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of SNRPB variant 1 (SEQ ID NO: 6) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8).

[0380] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of SNRPB variant 1 (SEQ ID NO: 6), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO: 9). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0381] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRBP variant 1 (SEQ ID NO: 10). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6) and a 3'UTR comprising the 3'UTR of SNRBP variant 1 (SEQ ID NO: 10). In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRBP variant 2 (SEQ ID NO: 11). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6) and a 3'UTR comprising the 3'UTR of SNRBP variant 2 (SEQ ID NO: 11).

[0382] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of gene XBG (SEQ ID NO: 12). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the SNRBP variant 1 gene (SEQ ID NO: 6) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12).

[0383] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of gene WPRE (SEQ ID NO: 13). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of SNRBP variant 1 (SEQ ID NO: 6) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0384] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the XBG gene (SEQ ID NO:7), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBA2 gene (SEQ ID NO:8). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the XBG gene (SEQ ID NO: 7) and a 3'UTR comprising the 3'UTR of the HBA2 gene (SEQ ID NO: 8).

[0385] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the XBG gene (SEQ ID NO: 7), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the HBB gene (SEQ ID NO: 9). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the XBG gene (SEQ ID NO: 7) and a 3'UTR comprising the 3'UTR of the HBB gene (SEQ ID NO: 9).

[0386] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the XBG gene (SEQ ID NO: 7), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRPB variant 1 (SEQ ID NO: 10). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the XBG gene (SEQ ID NO: 7) and a 3'UTR comprising the 3'UTR of the SNRPB variant 1 gene (SEQ ID NO: 10).

[0387] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the XBG gene (SEQ ID NO:7), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of SNRPB variant 2 (SEQ ID NO:11). In certain embodiments, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the XBG gene (SEQ ID NO: 7) and a 3'UTR comprising the 3'UTR of SNRPB variant 2 (SEQ ID NO: 11).

[0388] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the XBG gene (SEQ ID NO: 7), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene XBG (SEQ ID NO: 12). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the XBG gene (SEQ ID NO: 7) and a 3'UTR comprising the 3'UTR of the gene XBG (SEQ ID NO: 12).

[0389] In some embodiments, the polynucleotide comprises a 5'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 5'UTR of the XBG gene (SEQ ID NO: 7), and the polynucleotide comprises a 3'UTR having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to the 3'UTR of the gene WPRE (SEQ ID NO: 13). In a particular embodiment, the polynucleotide comprises a 5'UTR comprising the 5'UTR of the XBG gene (SEQ ID NO: 7) and a 3'UTR comprising the 3'UTR of the gene WPRE (SEQ ID NO: 13).

[0390] In various embodiments, the 5'UTR further comprises a eukaryotic initiation factor (eIF) recruitment sequence. In some embodiments, the eIF recruitment sequence comprises an eIF4G recruitment sequence. In some embodiments, the eIF4G recruitment sequence comprises APT17. In some embodiments, APT17 has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 14. In some specific embodiments, APT17 comprises the sequence of SEQ ID NO: 14.

[0391] In various embodiments, the 5' UTR does not form a stable secondary sequence structure containing the heterologous protein start codon, e.g., in some embodiments, the 5' UTR does not form a stable secondary sequence structure containing the heterologous protein start codon with a change in free energy (ΔG) of less than about -10 kcal / mol to about -80 kcal / mol.

[0392] In various embodiments, the 5'UTR is between about 30 nucleotides and about 250 nucleotides in length. In some embodiments, the 5'UTR is between about 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, or 75 nucleotides. , 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, 80 nucleotides, 81 nucleotides, 82 nucleotides, 83 nucleotides, 84 nucleotides, 85 nucleotides, 86 nucleotides, 87 nucleotides, 88 nucleotides, 89 nucleotides, 90 nucleotides, 91 nucleotides, 92 nucleotides, 93 nucleotides, 94 nucleotides, 95 nucleotides, 96 nucleotides, 97 nucleotides, 98 nucleotides, 99 nucleotides, 100 nucleotides, 101 nucleotides, 102 nucleotides, 103 nucleotides, 104 nucleotides, 105 nucleotides, 106 nucleotides, 107 nucleotides, 108 nucleotides, 109 nucleotides, 110 nucleotides, 111 nucleotides, 112 nucleotides, 113 nucleotides, 114 nucleotides, 115 nucleotides, 116 nucleotides, 117 nucleotides, 118 nucleotides, 119 nucleotides, 120 nucleotides, 121 nucleotides, 122 nucleotides, 123 nucleotides, 124 nucleotides, 125 nucleotides,126 nucleotides, 127 nucleotides, 128 nucleotides, 129 nucleotides, 130 nucleotides, 131 nucleotides, 132 nucleotides, 133 nucleotides, 134 nucleotides, 135 nucleotides, 136 nucleotides, 137 nucleotides, 138 nucleotides, 139 nucleotides, 140 nucleotides, 141 nucleotides, 142 nucleotides, 143 nucleotides, 144 nucleotides, 145 nucleotides, 146 nucleotides, 147 nucleotides, 148 nucleotides, 149 nucleotides, 150 nucleotides, 151 nucleotide, 152 nucleotide, 153 nucleotide, 154 nucleotide, 155 nucleotide, 156 nucleotide, 157 nucleotide, 158 nucleotide, 159 nucleotide, 160 nucleotide, 161 nucleotide, 162 nucleotide, 163 nucleotide, 164 nucleotide, 165 nucleotide, 166 nucleotide, 167 nucleotide, 168 nucleotide, 169 nucleotide, 170 nucleotide, 171 nucleotide, 172 nucleotide, 173 nucleotide, 174 nucleotide, 175 nucleotide, 176 nucleotides, 177 nucleotides, 178 nucleotides, 179 nucleotides, 180 nucleotides, 181 nucleotides, 182 nucleotides, 183 nucleotides, 184 nucleotides, 185 nucleotides, 186 nucleotides, 187 nucleotides, 188 nucleotides, 189 nucleotides, 190 nucleotides, 191 nucleotides, 192 nucleotides, 193 nucleotides, 194 nucleotides, 195 nucleotides, 196 nucleotides, 197 nucleotides, 198 nucleotides, 199 nucleotides, 200 nucleotides, 201 nucleotide, 202 nucleotide, 203 nucleotide, 204 nucleotide, 205 nucleotide, 206 nucleotide, 207 nucleotide, 208 nucleotide, 209 nucleotide, 210 nucleotide, 211 nucleotide, 212 nucleotide, 213 nucleotide, 214 nucleotide, 215 nucleotide, 216 nucleotide, 217 nucleotide, 218 nucleotide, 219 nucleotide, 220 nucleotide, 221 nucleotide, 222 nucleotide, 223 nucleotide, 224 nucleotide, 225 nucleotide,226 nucleotides, 227 nucleotides, 228 nucleotides, 229 nucleotides, 230 nucleotides, 231 nucleotides, 232 nucleotides, 233 nucleotides, 234 nucleotides, 235 nucleotides, 236 nucleotides, 237 nucleotides, 238 nucleotides, 239 nucleotides, 240 nucleotides, 241 nucleotides, 242 nucleotides, 243 nucleotides, 244 nucleotides, 245 nucleotides, 246 nucleotides, 247 nucleotides, 248 nucleotides, 249 nucleotides, 250 nucleotides, 251 nucleotides, 252 nucleotides, 252 nucleotides, 253 nucleotides, 254, or 255 nucleotides in length.

[0393] In certain embodiments, the 5'UTR further comprises an internal ribosome entry site (IRES).The internal ribosome entry site (IRES) element is a cis-acting RNA region that promotes the internal initiation of protein synthesis using a cap-independent mechanism.The specific types of IRES elements present in the genomes of various RNA viruses can perform the same function despite lacking sequence and secondary RNA structure conservation.Similarly, IRES elements may differ in the host factor requirements for recruiting ribosomal subunits.

[0394] In some embodiments, the 3'UTR has fewer than about three AU-rich regions (AREs). In certain embodiments, the 3'UTR has two AREs. In some other embodiments, the 3'UTR has one ARE. In still other embodiments, the UTR has no AREs. In some embodiments, the AU-rich region is a class I ARE. In some embodiments, the AU-rich region is a class II ARE. In still other embodiments, the AU-rich region is a class III ARE. Class I ARE elements, like the c-fos gene, have AUUUA motifs dispersed within or near U-rich regions. Class II elements, like the GM-CSF gene, have AUUUA motifs stacked within or near U-rich regions. Class III elements, like the c-jun gene, are a much less well-defined class that have U-rich regions but no AUUUA repeats.

[0395] An mRNA polynucleotide can contain a poly(A) tail or poly(A) sequence for nuclear export, translation, and stability of the mRNA. Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically messenger RNA (mRNA). The poly(A) tail consists of a stretch of RNA containing only adenine bases.

[0396] In some embodiments, the polynucleotide comprises a modification to the coding sequence of a heterologous protein to reduce ribosomal stacking or stalling during protein translation of the coding sequence, the modification comprising changing one or more three-base codons in the coding sequence that promote ribosomal stalling to three-base codons that reduce ribosomal stalling, thereby reducing ribosomal stalling or stacking during protein translation of the heterologous protein. Ribosomal stalling or stacking may be reduced by at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 90%, or 100%, as measured by standard methods in the art. In some embodiments, the modification does not change the amino acid sequence of the heterologous protein. In certain embodiments, the modification comprises modifying a codon encoding amino acid position 3, 4, 5, 6, 7, 8, 9, or 10 of the coding sequence to reduce ribosomal stalling or stacking. In some embodiments, the modification comprises modifying the codons encoding amino acid positions 3, 4, and 5 of the coding sequence to reduce ribosomal stalling or stacking.

[0397] In various embodiments, the polynucleotide further comprises a modification to the coding sequence of the heterologous protein to reduce the thymidine or uridine content of the coding sequence, wherein the modification does not change the amino acid sequence of the heterologous protein.

[0398] In some embodiments, the modification involves changing a first codon containing thymidine or uridine that encodes an amino acid to an alternative codon containing fewer thymidine or uridine than the first codon, where the modification does not alter the amino acid sequence of the heterologous protein. In some embodiments, the modification involves changing a first three-base codon containing thymidine or uridine that encodes an amino acid to an alternative three-base codon that does not have a thymidine or uridine content, where the modification does not alter the amino acid sequence of the heterologous protein. In various embodiments, the modification results in a 10% to 90% reduction in the thymidine or uridine content in the coding sequence, where the modification does not alter the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a 20% to 80% reduction in the thymidine or uridine content in the coding sequence, where the modification does not alter the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a 30% to 70% reduction in the thymidine or uridine content in the coding sequence, where the modification does not alter the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a 40%-60% reduction in the thymidine or uridine content in the coding sequence, and the modification does not change the amino acid sequence of the heterologous protein. In some embodiments, the modification results in an approximately 50% reduction in the thymidine or uridine content in the coding sequence, and the modification does not change the amino acid sequence of the heterologous protein. In certain embodiments, the modification results in an approximately 40% reduction in the thymidine or uridine content in the coding sequence, and the modification does not change the amino acid sequence of the heterologous protein. In some embodiments, the first three-base codon is modified to remove one, two, or three thymidine and / or uridine bases without changing the amino acid encoded by the codon.

[0399] In various embodiments, the polynucleotide further comprises a modification to the coding sequence of the heterologous protein that increases the GC content without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification comprises changing a first triplet codon containing a guanine or cytosine encoding an amino acid to an alternative triplet codon that has more guanines or cytosines than the first triplet codon, where the modification does not change the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 30% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 35% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 40% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 45% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 50% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 55% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 60% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 65% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 70% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 75% in the coding sequence without changing the amino acid sequence of the heterologous protein. In some embodiments, the modification results in a CG content of at least 80% in the coding sequence without changing the amino acid sequence of the heterologous protein. In various embodiments, the modification is a codon optimization process, which can be implemented, for example, by an algorithm or software.

[0400] In various embodiments, the heterologous protein comprises an NLS. In some embodiments, the NLS is located at the N-terminus of the heterologous protein. In other embodiments, the NLS is located at the C-terminus of the heterologous protein. In some embodiments, the heterologous protein comprises an NLS at the N-terminus of the heterologous protein and an identical NLS at the C-terminus. In other embodiments, the heterologous protein comprises an NLS at the N-terminus of the heterologous protein and a different NLS at the C-terminus. The NLS is selected from, but not limited to, SV40 NLS (SEQ ID NO: 15 or 19), NLS5 (SEQ ID NO: 16 or 20), CMYC NLS (SEQ ID NO: 17), or SV40H2 NLS (SEQ ID NO: 18). In some embodiments, the NLS comprises an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 15-20. In some embodiments, the NLS comprises the amino acid sequence of any one of SEQ ID NOs: 15-20.

[0401] In various embodiments, heterologous protein is engineered nuclease.In the present invention, any engineered nuclease can be used for the targeted insertion of donor template, including engineered meganuclease, zinc finger nuclease, TALEN, compact TALEN, CRISPR system nuclease or megaTAL.Engineered nuclease can cause the indel mutation of the chromosomal DNA of host cell.

[0402] In some particular embodiments, the polynucleotide comprises a 5' UTR having at least about 95% sequence identity to SEQ ID NO: 7 and a UTR Kozak sequence according to any one of SEQ ID NOs: 50-149, wherein the 5' UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and have at least 85% sequence identity to SEQ ID NO: 15, the coding sequence of the heterologous protein has been modified to reduce thymine or uracil content, and the 3' UTR has at least about 95% sequence identity to SEQ ID NO: 9, and the 3' UTR does not comprise any AREs.

[0403] In some particular embodiments, the polynucleotide comprises a 5' UTR having at least about 95% sequence identity to SEQ ID NO:1 and a UTR Kozak sequence according to any one of SEQ ID NOs:50-149, wherein the 5' UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and have at least 85% sequence identity to SEQ ID NO:15, the coding sequence of the heterologous protein has been modified to have reduced thymidine or uridine content, and the 3' UTR has at least about 95% sequence identity to SEQ ID NO:10, and the 3' UTR does not comprise any AREs.

[0404] In some particular embodiments, the polynucleotide comprises a 5' UTR having at least about 95% sequence identity to SEQ ID NO:2 and a UTR Kozak sequence according to any one of SEQ ID NOs:50-149, wherein the 5' UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and have at least 85% sequence identity to SEQ ID NO:15, the coding sequence of the heterologous protein has been modified to have reduced thymidine or uridine content, the 3' UTR has at least about 95% sequence identity to SEQ ID NO:10, and the 3' UTR does not comprise any AREs.

[0405] In some particular embodiments, the polynucleotide comprises a 5' UTR having at least about 95% sequence identity to SEQ ID NO:4 and a UTR Kozak sequence according to any one of SEQ ID NOs:50-149, wherein the 5' UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and have at least 85% sequence identity to SEQ ID NO:15, the coding sequence of the heterologous protein has been modified to reduce thymine or uracil content, the 3' UTR has at least about 95% sequence identity to SEQ ID NO:10, and the 3' UTR does not comprise any AREs.

[0406] In some particular embodiments, the polynucleotide comprises a 5' UTR having at least about 95% sequence identity to SEQ ID NO: 7 and a UTR Kozak sequence according to any one of SEQ ID NOs: 50-149, wherein the 5' UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and have at least 85% sequence identity to SEQ ID NO: 15, the coding sequence of the heterologous protein has been modified to reduce thymine or uracil content, the 3' UTR has at least about 95% sequence identity to SEQ ID NO: 10, and the 3' UTR does not comprise any AREs.

[0407] In some particular embodiments, the polynucleotide comprises a 5' UTR having at least about 95% sequence identity to SEQ ID NO:7 and a UTR Kozak sequence according to any one of SEQ ID NOs:50-149, wherein the 5' UTR does not comprise an upstream uATG sequence or an upstream open reading frame sequence, the heterologous protein is an engineered nuclease comprising a first NLS at the N-terminus and a second NLS at the C-terminus of the engineered nuclease, wherein the first NLS and the second NLS are identical and have at least 85% sequence identity to SEQ ID NO:15, the coding sequence of the heterologous protein has been modified to reduce thymine or uracil content, and the 3' UTR has at least about 95% sequence identity to SEQ ID NO:8, and the 3' UTR does not comprise any AREs.

[0408] In various embodiments, the polynucleotide is an mRNA. In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the 5' cap comprises a 5' methylguanosine cap. In some embodiments, the uridine present in the mRNA is pseudouridine or 2-thiouridine. In other embodiments, the uridine present in the mRNA is methylated. In some embodiments, the uridine present in the mRNA is N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

[0409] Further provided herein are recombinant DNA constructs comprising the polynucleotide. In some embodiments, the recombinant construct encodes a recombinant virus comprising the polynucleotide. Such viruses are known in the art and include recombinant retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV) (reviewed in Vannucci, et al. (New Microbiol. 2013, 36:1-22)). AAV useful in the present invention can have any serotype that allows for viral transduction into a target cell type and expression of heterologous proteins in the target cell. In certain embodiments, the AAV has the AAV2 or AAV6 serotype. AAV may be single-stranded, or alternatively, may be self-complementary so as not to require second-strand DNA synthesis in the host cell (McCarty, et al., Gene Ther., 2001, 8:1248-54).

[0410] A polynucleotide comprising a nucleic acid sequence encoding a heterologous protein can be delivered in DNA form (e.g., a plasmid) and / or via a virus (e.g., AAV). In some embodiments, the nucleic acid sequence encoding the protein can be operably linked to a promoter. In various embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the heterologous protein. As used herein, "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional linkage that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or discontinuous. When used to refer to the linkage of two polypeptide coding regions, operably linked is intended to mean that the coding regions are in the same reading frame. The cassette may further comprise at least one additional gene that is co-transformed into the organism. Alternatively, the additional genes may be provided on multiple expression cassettes. Such expression cassettes comprise multiple restriction and / or recombination sites for insertion of the polynucleotide under the transcriptional control of the regulatory region. The expression cassette may further comprise a selectable marker gene.

[0411] Numerous promoters can be used in the practice of the present invention. The promoter can be selected based on the desired results. The coding sequence can be combined with a constitutive promoter, a tissue-specific promoter, an inducible promoter, or other promoter for expression in host cells. For example, the constitutive promoter can be selected from the list of, but not limited to, T7AG, SV40, CMV, UBC, EF1A, PGK, ACTB, EF1α, PGK, UbC, and CAGG promoters (Norman et al., PLoS ONE, 2010, 5(8):e12413; Qin et al., PLoS ONE, 2010, 5(5):e10611). In some embodiments, the promoter can be a viral promoter, such as an endogenous promoter derived from a virus (e.g., the LTR of a lentiviral vector). In a preferred embodiment, the heterologous polypeptide coding sequence is operably linked to a promoter that preferentially promotes gene expression in target cells. In some examples, the heterologous polypeptide coding sequence is operably linked to a synthetic promoter, such as the JeT promoter (US Pat. No. 6,555,674).

[0412] In some embodiments, the polynucleotide is delivered via a vector, such as a plasmid. Various plasmids can be used in the present invention. For example, the plasmid may have a nucleic acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, of any one of SEQ ID NOS: 21-49. In certain embodiments, the plasmid vector may be any one of SEQ ID NOS: 21-49.

[0413] Further provided herein are lipid particles comprising polynucleotides. In some embodiments, the lipid particles are lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise a polynucleotide that is mRNA. In some embodiments, the polynucleotide encodes an engineered nuclease. As used herein, the term "lipid nanoparticle" refers to a lipid composition having a typically spherical structure with an average diameter of 10 to 1000 nanometers. In some formulations, the lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use in the present invention.

[0414] Also provided herein are eukaryotic cells comprising the polynucleotides, wherein the protein level of the encoded heterologous protein in the eukaryotic cells comprising the polynucleotides is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to control cells. In the event that the polynucleotide is mRNA, the half-life of the polynucleotide in a eukaryotic cell containing the polynucleotide is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to a control cell. In the event that the polynucleotide is DNA, the half-life of mRNA produced from the polynucleotide in a eukaryotic cell containing the polynucleotide is increased by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to a control cell.

[0415] In various embodiments, the polynucleotide encodes an engineered nuclease, and protein levels of the encoded engineered nuclease in eukaryotic cells containing the polynucleotide are increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to control cells. In the event that the polynucleotide is mRNA, the half-life of the polynucleotide in a eukaryotic cell containing the polynucleotide is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to a control cell. In the event that the polynucleotide is DNA, the half-life of mRNA produced from the polynucleotide in a eukaryotic cell containing the polynucleotide is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to a control cell.

[0416] The eukaryotic cells containing the polynucleotide have a genome editing efficiency that is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more, compared to control cells. In various embodiments, genome editing efficiency is measured by the percentage of indels.

[0417] V. Methods for Expressing Heterologous Proteins

[0418] Provided herein is a method for expressing heterologous protein in eukaryotic cells, comprising introducing a polynucleotide into eukaryotic cells so that the heterologous protein is expressed in the cells.In various embodiments, the polynucleotide is a recombinant DNA construct as disclosed elsewhere herein.The polynucleotide can be introduced into eukaryotic cells by lipid nanoparticles, recombinant viruses, or any other means for introducing polynucleotides into cells.In some embodiments, the polynucleotide is introduced into eukaryotic cells by a recombinant virus, which is any one of recombinant adenovirus, recombinant lentivirus, recombinant retrovirus, or recombinant adeno-associated virus.In some embodiments, the heterologous protein is an engineered nuclease, which is expressed in eukaryotic cells, and the genome editing efficiency increases in the cells compared with control cells.

[0419] In some embodiments, the protein level of the heterologous protein in the eukaryotic cell is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000% or more compared to control cells, or, in the event that the polynucleotide is mRNA, by at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 13-fold, 14-fold, 15-fold or more compared to control cells. Similarly, the half-life of an mRNA polynucleotide in a eukaryotic cell is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000% or more, or by at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 13-fold, 14-fold, 15-fold or more, as compared to a control cell. In the event that the polynucleotide is DNA, the half-life of mRNA produced from the DNA polynucleotide in a eukaryotic cell containing the polynucleotide is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000% or more, or by at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 13-fold, 14-fold, 15-fold or more, as compared to a control cell.

[0420] In certain embodiments, mRNA persistence is increased by about 2-10 fold in eukaryotic cells compared to control eukaryotic cells. For example, mRNA persistence can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000% or more compared to control cells, or by at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 13-fold, 14-fold, 15-fold or more compared to control cells. The mRNA polynucleotides disclosed herein can persist in eukaryotic cells for about 1 hour to about 96 hours. In some embodiments, the mRNA remains in the cell for about 8 hours to about 48 hours. In certain embodiments, the mRNA remains in the cell for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 24 hours, 25 hours, 30 hours, 35 hours, 36 hours, 40 hours, 45 hours, 48 ​​hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 72 hours, 75 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, 105 hours, 110 hours, or more. In some embodiments, the mRNA remains in the cell for at least 8 hours. In some embodiments, the mRNA remains in the cell for at least 24 hours.

[0421] Also provided herein is a method for treating a disease in a subject in need thereof, comprising administering a therapeutically effective amount of a polynucleotide encoding a heterologous protein disclosed herein. In some embodiments, the disease is a genetic disease. In some embodiments, the heterologous protein is an engineered nuclease. The engineered nuclease can induce indel mutations in a subject so as to correct a genetic mutation associated with the genetic disease and / or to alleviate or ameliorate the symptoms caused by the genetic disease. Any engineered nuclease can be used in the method for treating a disease. For example, engineered nucleases include, but are not limited to, engineered meganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPR system nucleases, or megaTALs.

[0422] In some embodiments, the method of treating a disease comprises topical administration of a pharmaceutical composition described herein to a subject in need thereof. In some other embodiments, the method of treating a disease comprises intravenous injection or infusion of a pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the administration of the pharmaceutical composition is completed instantaneously. In some embodiments, the topical administration of the pharmaceutical composition is completed instantaneously. In some embodiments, the topical administration of the pharmaceutical composition is completed over a process of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some embodiments, the intravenous injection of the pharmaceutical composition is completed instantaneously. In some embodiments, the intravenous infusion of the pharmaceutical composition is completed over a process of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes.

[0423] In some embodiments of the treatment method, therapeutic protein is a peptide or protein as part of vaccine, antibody, engineered nuclease, RNA modifying enzyme or DNA modifying enzyme.In certain embodiments, therapeutic protein is engineered nuclease.In some embodiments, engineered nuclease is engineered meganuclease, TALEN, zinc finger nuclease, CRISPR system nuclease, compact TALEN or megaTAL, as described elsewhere herein.

[0424] VI. Pharmaceutical Compositions

[0425] Also provided herein are pharmaceutical compositions comprising the polynucleotide. Such pharmaceutical compositions can be prepared by known techniques. In some embodiments, the pharmaceutical composition comprises a polynucleotide encoding a heterologous protein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a DNA construct comprising a polynucleotide encoding a heterologous protein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises a recombinant virus comprising a polynucleotide encoding a heterologous protein and a pharmaceutically acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and not harmful to the subject. In some embodiments, the pharmaceutical composition used in the methods and compositions disclosed herein can further comprise one or more additional agents useful for treating a disease in the subject.

[0426] In some embodiments, the pharmaceutical composition comprises a recombinant virus comprising a polynucleotide encoding a heterologous protein described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition contains 1.0 x 10 per milliliter of the recombinant virus. 11 ~1.0×10 13 In some embodiments, the pharmaceutical composition comprises an AAV vector genome at a concentration of 1.0 x 10 per milliliter. 11 ~1.0×10 13In some embodiments, the pharmaceutical composition comprises a recombinant adeno-associated virus at a concentration of 1.0 x 10 vector genome per milliliter. 11 ~1.0×10 13 In some embodiments, the pharmaceutical composition comprises a recombinant retrovirus at a concentration of 1.0 x 10 vector genome per milliliter. 11 ~1.0×10 13 In some embodiments, the pharmaceutical composition comprises a recombinant lentivirus at a concentration of 1.0 x 10 vector genome per milliliter. 11 ~1.0×10 13 It contains recombinant adenovirus at the concentration of the vector genome.

[0427] In some embodiments, a pharmaceutical composition comprises a heterologous protein polynucleotide that is mRNA and a pharmaceutically acceptable carrier. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.1 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.2 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.3 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.4 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.5 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.6 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.7 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.8 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 0.9 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 1.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 2.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 3.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 4.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 5.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 6.0 mg / ml.In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 7.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 8.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 9.0 mg / ml. In some embodiments, a composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration of at least 10.0 mg / ml. A composition comprising mRNA encoding a heterologous protein comprises the mRNA at a concentration ranging from 0.1 mg / ml to 10.0 mg / ml.

[0428] In some embodiments, the pharmaceutical composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.1 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.2 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.3 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.4 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.5 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.6 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.7 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector (at least about 0.8 mg / ml) having a polynucleotide encoding a heterologous protein. In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 0.9 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 1.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 2.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 3.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 4.0 mg / ml).In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 5.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 6.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 7.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 8.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 9.0 mg / ml). In some embodiments, the composition comprises a recombinant DNA vector comprising a polynucleotide encoding a heterologous protein (at least about 10.0 mg / ml).

[0429] As the terms "effective amount" or "therapeutic amount" are used herein, the exact amount to be administered can be determined by a physician taking into account individual differences in age, weight, disease state, tumor size (if present), extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a subject receives approximately 1 x 10 11 ~Approx. 1×10 13 A pharmaceutical composition comprising a vector genome dose of the recombinant virus of the present disclosure can be administered in a volume of 1 ml. In certain embodiments, a subject receives about 1 x 10 11 ~Approx. 1×10 13 A pharmaceutical composition comprising a vector genome dose of the recombinant virus of the present disclosure may be administered in a volume of 2 ml. In certain embodiments, a subject receives approximately 1 x 10 11 ~Approx. 1×10 13 A pharmaceutical composition comprising a vector genome dose of the recombinant virus of the present disclosure may be administered in a volume of 3 ml. In certain embodiments, a subject receives approximately 1 x 10 11 ~Approx. 1×10 13 A pharmaceutical composition comprising a vector genome dose of the recombinant virus of the present disclosure may be administered in a volume of 4 ml. In certain embodiments, a subject receives approximately 1 x 1011 ~Approx. 1×10 13 A pharmaceutical composition containing a vector genome dose of the recombinant virus of the present disclosure can be administered in a volume of 5 ml. The optimal dosage and treatment regimen for a particular patient can be easily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0430] In certain embodiments, the pharmaceutical composition comprising the mRNA comprises about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, About 25mg, about 26mg, about 27mg, about 28mg, about 29mg, about 30mg, about 31mg, about 32mg, about 33mg, about 34mg, about 35mg, about 36mg, about 37mg, about 3 8mg, about 39mg, about 40mg, about 41mg, about 42mg, about 43mg, about 44mg, about 45mg, about 46mg, about 47mg, about 48mg, about 49mg, about 50mg, about 51m g, about 52mg, about 53mg, about 54mg, about 55mg, about 56mg, about 57mg, about 58mg, about 59mg, about 60mg, about 61mg, about 62mg, about 63mg, about 64mg , about 65mg, about 66mg, about 67mg, about 68mg, about 69mg, about 70mg, about 71mg, about 72mg, about 73mg, about 74mg, about 75mg, about 76mg, about 77mg, about The subject is administered a dose containing 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg of mRNA. The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0431] In certain embodiments, the pharmaceutical composition comprising the recombinant DNA vector is administered in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, 4mg, about 25mg, about 26mg, about 27mg, about 28mg, about 29mg, about 30mg, about 31mg, about 32mg, about 33mg, about 34mg, about 35mg, about 36mg, about 37mg , about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 5 1mg, about 52mg, about 53mg, about 54mg, about 55mg, about 56mg, about 57mg, about 58mg, about 59mg, about 60mg, about 61mg, about 62mg, about 63mg, about 64mg , about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 7 The dosage of the DNA vector is administered to the subject at a dose of 8 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg or about 100 mg. The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the medical field by monitoring the patient for symptoms of disease and adjusting treatment accordingly.

[0432] In certain embodiments, pharmaceutical compositions comprising the polynucleotides of the present disclosure can be administered by intravenous delivery in a single dose. In certain embodiments, intravenous delivery in a single dose can be a one-time treatment. Intravenous delivery in a single dose can provide sustained relief to subjects with genetic diseases and / or associated symptoms. Relief can include, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 A number of minutes, such as 6, 57, 58, 59, or more than 59 minutes, including but not limited to, 1, 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, or more than 48 hours, including but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days, or more than 31 days, including but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days The polynucleotides of the present disclosure may be administered for several weeks, such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 months, or for several years, such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 years. In other embodiments, pharmaceutical compositions comprising polynucleotides of the present disclosure may be administered by intravenous delivery in multiple doses. [Example]

[0433] The present invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed by the claims that follow the examples below.

[0434] Example 1

[0435] Effect of the ribosome recruitment sequence APT17 on editing of the engineered meganuclease recognition sequence HAO 1-2 in human cell lines

[0436] 1. Methods and Materials

[0437] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of an engineered meganuclease designed to target a recognition sequence within the human HAO gene using an indel detection assay. The engineered meganuclease used in these experiments was HAO 1-2L.30S19, previously described in PCT Publication WO 2020 / 132659.

[0438] In these experiments, several different mRNA designs were tested to evaluate the effect of ribosome recruitment sequences on in vitro editing efficiency. Using the Lonza Amaxa 4D system, mRNA from Table 1 was electroporated into human cells (2 ng of HEK293). All coding sequences for meganucleases were further modified using alternative codon sequences to reduce uridine content while keeping the amino acid sequence identical. Each mRNA contained an N1-methylpseudouridine and a 7-methylguanosine cap. The recruitment sequence-only mRNA had the recruitment sequence linked to a Kozak sequence (GGCCCCATGGC, SEQ ID NO: 145).

[0439] [Table 1]

[0440] Cells were harvested 2.5, 5, and 24 hours after electroporation for gDNA formulations and assessed for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency was greater than 90%. gDNA was prepared using the Macherey Nagel NucleoSpin Blood QuickPure kit.

[0441] Digital droplet PCR (Digital PCR) was used to determine the frequency of targeted insertions and deletions (indels%) in the HAO 1-2 recognition sequence using primers P1, F1, and R1, and primers P2, F2, and R2, to generate a reference amplicon. Amplification was multiplexed in a 20 μL reaction containing 1× ddPCR probe supermix (without dUTP, BioRad), 250 nM of each probe, 900 nM of each primer, 5 U of HindIII-HF, and approximately 50 ng of cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). The cycling conditions for HAO 1-2 were as follows: One cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 95°C (1°C / s ramp) for 30 seconds, 62°C (1°C / s ramp) for 30 seconds, 72°C (0.2°C / s ramp) for 2 minutes, one cycle of 98°C for 10 minutes, 4°C hold.

[0442] Droplets were analyzed using a QX200 droplet reader (BioRad), and data were acquired and analyzed using QuantaSoft analysis software (BioRad). Indel frequencies were calculated by dividing the number of positive copies of the binding site probe by the number of positive copies of the reference probe and comparing the loss of FAM+ copies in nuclease-treated cells with mock-transfected cells.

[0443] Primer set

[0444] P1:42 HAO1 2 BHQ 1 BS Probe: TTCCTCACCAATGTCTTGT FAM (SEQ ID NO: 150) F1: 28-HAO21-22 F2: CCACATAAGATTTGGCAAGCC (SEQ ID NO: 151) R1: 27-HAO21-22 R2: GGAAAAGAACGACACCCTTTG (SEQ ID NO: 152) P2:33 HAO23 / 24 P1 REF:CCCGGCTAATTTGTATCA VIC (SEQ ID NO: 153) F2:29-HAO23-24 f1:GCTCACTTGATGTAAGCAACAG (SEQ ID NO: 154) R2: 32-HAO23-24 R2: ACACACCACCAACGTAAAAC (SEQ ID NO: 155)

[0445] 2.Results

[0446] In these studies, indels (insertions and deletions) were measured by ddPCR across multiple time points. In HEK293 cells, control mRNA at a low mRNA dose of 2 ng showed indels ranging from 5% (2.5 h), 13% (5 h), and 37% (24 h). Indels in RS HBA2 mRNA ranged from 6%, 22%, and 55% across time points, while indels from HAO1-RS-only mRNA were 5%, 13%, and 36% at the same time points (Figure 1).

[0447] 3. Conclusion

[0448] These studies demonstrate the ability of improved mRNAs encoding engineered meganucleases to generate indels at the HAO 1-2 recognition sequence in human cell lines in vitro. We directly compared mRNAs encoding meganucleases containing recruitment sequence mutations with meganucleases targeting the HAO 1-2 site without the recruitment sequence. With a recruitment sequence linked to the UTR, RS HBA2 mRNA encoding the same HAO 1-2 nuclease exhibited higher editing efficiency at 5 and 24 hours in human cell lines than control or RS-only mRNAs, demonstrating that adding a ribosome recruitment sequence to mRNA can improve protein expression and concomitant gene editing efficiency.

[0449] Example 2

[0450] Effects of various UTRs on editing of engineered meganuclease recognition sequence F8R 17-18 in human cell lines

[0451] 1. Methods and Materials

[0452] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of an engineered meganuclease designed to target a recognition sequence within the human F8R gene by digital PCR using an indel detection assay. The engineered meganuclease used in these experiments was the F8R 17-18L.1.35 meganuclease previously described in PCT Publication WO 2019 / 089913.

[0453] In this experiment, mRNA encoding the F8R 17-18L1.35 meganuclease in Table 2 was electroporated into BNL C.2 cells (200 ng or 20 ng) using a Lonza Amaxa 4D system.

[0454] [Table 2]

[0455] Cells were harvested 24 hours after electroporation for gDNA formulations and assessed for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency was greater than 90%. gDNA was prepared using the Macherey Nagel NucleoSpin Blood QuickPure kit.

[0456] Digital PCR was used to determine the frequency of targeted insertions and deletions (indels%) in the F8R17-18 recognition sequence using primers P1, F1, and R1, and primer P2, to generate a reference. Amplification was performed in a 20-µL reaction containing 1x ddPCR probe supermix (without dUTP, BioRad), 250 nM of each probe, 900 nM of each primer, 5 U of HindIII-HF, and approximately 50 ng of cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). The cycling conditions for F8R17-18 were as follows: one cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 94°C (2°C / s ramp) for 30 seconds, 56°C (2°C / s ramp) for 30 seconds, and 72°C (2°C / s ramp) for 2 minutes, followed by one cycle of 98°C for 10 minutes and a 4°C hold.

[0457] Droplets were analyzed using a QX200 droplet reader (BioRad), and data were acquired and analyzed using QuantaSoft analysis software (BioRad). Indel frequencies were calculated by dividing the number of positive copies of the binding site probe by the number of positive copies of the reference probe and comparing the loss of HEX+ copies in nuclease-treated cells with mock-transfected cells.

[0458] Primer set

[0459] P1:720 F8R17-18 BS probe: CCTCCCAGGAGTACTTCTCCAGG HEX (SEQ ID NO: 156) F1:721 F8R17-18 FWD1 F1:GATGCCTTCAGTGTCCTT (SEQ ID NO: 157) R1:724 F8R17-18REV2 R1:CTTTGCTGACGTCCTAGT (SEQ ID NO: 158) P2:771 F8R17-18REF2 probe: TACACGGGACACCTCACACCTG FAM (SEQ ID NO: 159)

[0460] 2.Results

[0461] In these studies, indels (insertions and deletions) were measured by ddPCR at 24 hours. In BNL C.2 cells at 200 ng or 20 ng of mRNA, F8R17-18L1.35 HBA2 at the high mRNA dose showed >60% indels at 24 hours. F8R17-18L1.35 HSD17B4 had >50% indels at 24 hours, and F8R17-18L1.35 MOD had >55% indels. F8R17-18L1.35 HBA2 at the low mRNA dose showed >25% indels at 24 hours. F8R17-18L1.35 HSD17B4 had 25% indels at 24 hours, and F8R17-18L1.35 MOD had <20% indels (Figure 2).

[0462] 3. Conclusion

[0463] These studies demonstrate the ability of the F8R17-18 meganuclease to generate indels at the F8R17-18 recognition sequence in vitro. mRNAs encoding meganucleases containing mutations in the 5' UTR sequence of either HSD17B4 or MOD were directly compared with control mRNAs containing the 5' HBA2 UTR and 3' WPRE UTR. In all cases, at high or low mRNA doses, the control mRNA had higher or similar editing efficiency than the other UTR combinations. These results indicated that these tested UTR combinations were not superior to the combination of the 5' HBA2 UTR and 3' WPRE UTR.

[0464] Example 3

[0465] Effects of various UTRs on editing of engineered meganuclease recognition sequences HAO 1-2 in human cell lines

[0466] 1. Methods and Materials

[0467] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of an engineered meganuclease designed to target a recognition sequence within the human HAO gene by digital PCR using an indel detection assay. The engineered meganuclease used in these experiments was HAO 1-2L.30S19, previously described in PCT Publication WO 2020 / 132659.

[0468] In these experiments, mRNA encoding the HAO1-2L.30 S19 meganuclease from Table 3, testing various 5' and 3' UTR combinations, was electroporated into human cells (HEP3B, 2 ng) using a Lonza Amaxa 4D system. All coding sequences for the meganucleases were further modified using alternative codon sequences to reduce uridine content while keeping the amino acid sequence identical. Each mRNA contained an N1-methylpseudouridine and a 7-methylguanosine cap.

[0469] [Table 3]

[0470] Cells were harvested 1 and 2 days after electroporation for gDNA preparations, or 2 days only, and assessed for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency exceeded 90%. Cells were harvested at two additional time points between 6 and 9 days after electroporation for gDNA extraction. gDNA was prepared using the Macherey Nagel NucleoSpin Blood QuickPure kit.

[0471] Digital PCR was used to determine the frequency of targeted insertions and deletions (indels%) in the HAO1-2 recognition sequence using primers P1, F1, and R1, and primers P2, F2, and R2, to generate a reference amplicon outside the HAO1-2 recognition sequence (OFF amplicon ddPCR). Additionally, another digital PCR was used to determine the frequency of targeted insertions and deletions (indels%) in the HAO1-2 recognition sequence using primers P1, F1, R1, and P3. In this ddPCR, primer P3 was used as an internal amplicon reference (ON amplicon ddPCR). Amplifications were multiplexed in 20 μL reactions containing 1× ddPCR probe supermix (without dUTP, BioRad), 250 nM of each probe, 900 nM of each primer, 5 U of HindIII-HF, and approximately 50 ng of cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). The cycling conditions for HAO 1-2 (OFF) were: 1 cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 95°C (1°C / s ramp) for 30 seconds, 62°C (1°C / s ramp) for 30 seconds, and 72°C (0.2°C / s ramp) for 2 minutes, 1 cycle of 98°C for 10 minutes, and a 4°C hold. The cycling conditions for HAO 1-2 (ON) were: 1 cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 95°C (1°C / s ramp) for 30 seconds, 61°C (1°C / s ramp) for 30 seconds, and 72°C (0.2°C / s ramp) for 2 minutes, 1 cycle of 98°C for 10 minutes, and a 4°C hold.

[0472] Droplets were analyzed using a QX200 droplet reader (BioRad), and data were acquired and analyzed using QuantaSoft analysis software (BioRad). Indel frequencies were calculated by dividing the number of positive copies of the binding site probe by the number of positive copies of the reference probe and comparing the loss of FAM+ copies in nuclease-treated cells with mock-transfected cells.

[0473] Primer set

[0474] P1:42 HAO1 2 BHQ 1 BS Probe: TTCCTCACCAATGTCTTGT FAM (SEQ ID NO: 150) F1: 28-HAO21-22 F2: CCACATAAGATTTGGCAAGCC (SEQ ID NO: 151) R1: 27-HAO21-22 R2: GGAAAAGAACGACACCCTTTG (SEQ ID NO: 152) P2:33 HAO23 / 24 P1 REF:CCCGGCTAATTTGTATCA VIC (SEQ ID NO: 153) F2:29-HAO23-24 f1:GCTCACTTGATGTAAGCAACAG (SEQ ID NO: 154) R2: 32-HAO23-24 R2: ACACACCACCAACGTAAAAC (SEQ ID NO: 155) P3:44 HAO12 Ref Probe 1: TGTGGTCACCCTCTGCACAGTGT HEX (SEQ ID NO: 160)

[0475] 2.Results

[0476] In Figure 3A, using the "ON" ddPCR indel assay, the HBA2 / WPRE control mRNA yielded approximately 10% to 15% indels from day 2 to day 9. XBG / XBG NLS5 mRNA and HBA2 / HBA2 mRNA performed similarly. In contrast, XBG / XBG SV40 (SEQ ID NO: 24) mRNA yielded indels ranging from greater than 20% to approximately 30% from day 2 to day 9. SNRPB V1 mRNA yielded indels ranging from approximately 15% to approximately 23% from day 2 to day 9, and SNRPB V2 mRNA yielded indels ranging from approximately 13% to approximately 18%. Similar results were obtained using the same tested mRNAs in Figures 3B, 3C, and 3D.

[0477] 3. Conclusion

[0478] These studies demonstrate the ability of the HAO1-2 meganuclease to generate indels at the HAO1-2 recognition sequence in human cell lines in vitro. MRNAs encoding meganucleases containing mutations in the 5' and 3' UTR sequences had increased indels compared to the control HBA2 / WPRE mRNA. The use of the NLS5 N-terminal NLS significantly reduced the percentage of detected indels at all time points, indicating that the SV40 NLS may be superior when used with engineered nucleases that require transfer to the nuclease to perform their function of cleaving DNA. Additionally, the addition of the SNRPBV2 3' UTR slightly reduced the percentage of indels. This finding may be due to the presence of an AU-rich region found in the 3' UTR.

[0479] Example 4

[0480] Effects of various UTRs on editing of engineered meganuclease recognition sequences HAO 1-2 in human cell lines

[0481] 1. Methods and Materials

[0482] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of an engineered meganuclease designed to target a recognition sequence within the human HAO gene by digital PCR using an indel detection assay. The engineered meganuclease used in these experiments was HAO 1-2L.30S19, previously described in PCT Publication WO 2020 / 132659.

[0483] In these experiments, mRNAs encoding HAO1-2L.30 S19 meganucleases with the additional variable 5' and 3' UTRs in Table 4 were electroporated into human cells (HEP3B, 2 ng) using a Lonza Amaxa 4D system. All coding sequences for the meganucleases were further modified using alternative codon sequences to reduce uridine content while keeping the amino acid sequence identical. Each mRNA contained an N1-methylpseudouridine and a 7-methylguanosine cap.

[0484] [Table 4]

[0485] Cells were harvested 2 days after electroporation for gDNA preparation and assessed for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency exceeded 90%. Cells were harvested at two additional time points between 6 and 9 days after electroporation for gDNA extraction. gDNA was prepared using the Macherey Nagel NucleoSpin Blood QuickPure kit.

[0486] Digital PCR to determine the frequency of targeted insertions and deletions (% indels) for both the "ON" and "OFF" assays was performed as described in Example 3.

[0487] 2.Results

[0488] In these studies, indels (insertions and deletions) were measured by ddPCR across multiple time points in HEP3B cells with 2 ng of mRNA using two biological replicates. Experimental data for the HAO1-2 "OFF" amplicon assay are listed in Table 5 and Figure 4A. Experimental data for the HAO1-2 "ON" amplicon assay are listed in Table 6 and Figure 4B. Data from this experiment were reconstructed to visualize any potential trends in 5' or 3' UTR selection, as shown in Figure 4C and Figure 4D.

[0489] [Table 5]

[0490] [Table 6]

[0491] 3. Conclusion

[0492] These studies demonstrate the ability of the HAO1-2 meganuclease to generate indels at the HAO1-2 recognition sequence in human cell lines in vitro. mRNAs encoding the HAO1-2 meganuclease containing mutations in the 5' and 3' UTR sequences were directly compared to control mRNAs containing the 5' HBA2 UTR and 3' WPRE UTR. In most cases, the control mRNA resulted in significantly lower editing efficiencies than mRNAs with the unique UTR combinations tested. Notably, the 5' HBA2 UTR and 3' XBG UTR performed significantly worse than the control mRNA. We observed that the SNRPB V1 3' UTR resulted in an increased percentage of indels in each of the paired 5' UTRs tested when compared with the use of the 3' XBG UTR (Figure 4C). Furthermore, in the case of the 5' UTR, XBG tends to generate higher indels than the 5' SNRPB V1 UTR. In addition, use of the 5'HBA2 UTR typically resulted in a significant decrease in activity.

[0493] Example 5

[0494] Effects of various UTRs on editing of engineered meganuclease recognition sequences HAO 1-2 in human cell lines across different doses

[0495] 1. Methods and Materials

[0496] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of an engineered meganuclease designed to target a recognition sequence within the human HAO gene by digital PCR using an indel detection assay. The engineered meganuclease used in these experiments was HAO 1-2L.30S19, previously described in PCT Publication WO 2020 / 132659.

[0497] In these experiments, mRNAs encoding HAO1-2L.30 S19 meganucleases with the additional variable 5' and 3' UTRs in Table 7 were electroporated into human cells (2 ng, 1 ng, 9.5 ng, and 0.25 ng HEP3B) using a Lonza Amaxa 4D system. Digital PCR to determine the frequency of targeted insertions and deletions (% indels) for both the "ON" and "OFF" assays was performed as described in Example 3. All coding sequences for the meganucleases were further modified using alternative codon sequences to reduce uridine content while keeping the amino acid sequence identical. Each mRNA contained an N1-methylpseudouridine and a 7-methylguanosine cap.

[0498] [Table 7]

[0499] 2.Results

[0500] In these studies, indels (insertions and deletions) were measured by ddPCR in HEP3B cells with multiple low doses of mRNA using two biological replicates. Experimental data for the HAO1-2 OFF amplicon assay are listed in Table 8 and shown in Figure 5A. Experimental data for the HAO1-2 ON amplicon assay are listed in Table 9 and shown in Figure 5B.

[0501] [Table 8]

[0502] [Table 9]

[0503] 3. Conclusion

[0504] These studies demonstrate the ability of mRNA containing the unique UTR combination encoding the HAO1-2 meganuclease to generate a higher percentage of indels than the control (HBA.WPRE) across all doses. This effect is maintained down to a low RNA dose of 0.25 ng, with ALB.SNRPB mRNA generating approximately four-fold more indels than the control mRNA.

[0505] Example 6

[0506] Editing the HAO 25-26 recognition sequence in human cell lines using improved mRNA encoding the engineered HAO 25-26 meganuclease

[0507] 1. Methods and Materials

[0508] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of engineered meganucleases designed to bind and cleave a target sequence (i.e., the HAO25-26 recognition sequence) within exon 2 of the HAO1 gene by digital PCR using an indel detection assay. The engineered meganucleases used in this experiment were the HAO25-26L.1128 and HAO25-26L.1434 meganucleases, encoded by the mRNAs of SEQ ID NOs: 46-49.

[0509] These studies were performed using an in vitro cell-based system and evaluated the editing efficiency of different HAO 25-26 meganucleases by digital PCR using an indel detection assay.

[0510] In these experiments, mRNAs utilizing a combination of the 5'ALB UTR and 3'SNRPB V1 UTR with an additional C-terminal NLS as part of the engineered meganuclease were tested against standard mRNAs utilizing the 5'HBA2 UTR and 3'WPRE UTR. The nucleic acid coding sequence of the meganuclease in the improved mRNAs was further modified using alternative codon sequences to reduce the uridine content while keeping the amino acid sequence identical. Each mRNA in the unmodified and improved mRNAs contained an N1-methylpseudouridine and 7-methylguanosine cap. Each meganuclease-encoding mRNA was electroporated into HepG2 cells at doses of 0.1 ng, 0.5 ng, 2 ng, 10 ng, 50 ng, and 100 ng using a Lonza Amaxa 4D system. The mRNAs tested in this experiment are listed in Table 10.

[0511] [Table 10]

[0512] Cells were harvested 7 days after electroporation for gDNA formulations and assessed for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency was greater than 90%. gDNA was prepared using a Macherey Nagel NucleoSpin Blood QuickPure kit.

[0513] Digital PCR was used to determine the frequency of targeted insertions and deletions (indels%) in the HAO 25-26 recognition sequence using primers P1, F1, and R1, and primers P2, F2, and R2, to generate a reference amplicon. Amplification was multiplexed in a 20 μL reaction containing 1× ddPCR probe supermix (without dUTP, BioRad), 250 nM of each probe, 900 nM of each primer, 5 U of HindIII-HF, and approximately 50 ng of cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). The cycling conditions for HAO 25-26 were as follows: The cycling conditions for HAO 3-4 were: 1 cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 94°C (1°C / s ramp) for 30 seconds, 62°C (1°C / s ramp) for 30 seconds, 72°C (0.2°C / s ramp) for 2 minutes, 1 cycle of 98°C for 10 minutes, 4°C hold. The cycling conditions for HAO 3-4 were: 1 cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 94°C (1°C / s ramp) for 30 seconds, 55°C (1°C / s ramp) for 30 seconds, 72°C (0.2°C / s ramp) for 2 minutes, 1 cycle of 98°C for 10 minutes, 4°C hold.

[0514] Droplets were analyzed using a QX200 droplet reader (BioRad), and data were acquired and analyzed using QuantaSoft analysis software (BioRad). Indel frequencies were calculated by dividing the number of positive copies of the binding site probe by the number of positive copies of the reference probe and comparing the loss of FAM+ copies in nuclease-treated cells with mock-transfected cells.

[0515] Primer set

[0516] P1: 34 HAO 25 / 26 P1 BS Probe: TTGGATACAGCTTCCATCTA FAM (SEQ ID NO: 161) F1: 21-HAO 25-25-15-16 F2: ACCAAACAAACAGTAAAATTGCC (SEQ ID NO: 162) R1:14-HAO15-16 25-26 R:GAGGTCGATAAACGTTAGCCTC (SEQ ID NO: 163) P2:44 12 Ref Probe 1: TGTGGTCACCCTCTGCACAGTGT HEX (SEQ ID NO: 164) F2: 28-HAO21-22 F2: CCACATAAGATTTGGCAAGCC (SEQ ID NO: 165) R2: 27-HAO21-22 R2: TGTGGTCACCCTCTGCACAGTGT (SEQ ID NO: 166)

[0517] 2.Results

[0518] In these studies, indels (insertions and deletions) were measured by ddPCR across multiple doses. The percentage of indels was significantly enhanced using modified mRNA constructs with alternative UTRs and uridine deletions. At a 10 ng dose, HAO25-26L.1128 meganuclease resulted in approximately 35% indel formation, while the modified construct designated "MAX" resulted in approximately 77% indel formation (Figure 6). Similarly, a 10 ng dose of HAO25-26L.1434 meganuclease resulted in approximately 33% indel formation, while the modified construct encoding HAO25-26L.1434 meganuclease designated "MAX" resulted in approximately 86% indels (Figure 6). The trend toward increased indel formation remained consistent across all doses, but the difference between the two types of mRNA decreased as the dose increased.

[0519] 3. Conclusion

[0520] These studies demonstrate the ability of the HAO 25-26 meganuclease to generate indels at the HAO 25-26 recognition sequence in HepG2 cells. The experiments further demonstrate that modifications to the mRNA encoding the meganuclease can significantly impact indel formation, resulting in much greater indel formation at lower mRNA doses. This has the advantage of reducing the amount of mRNA that needs to be delivered to target cells and potentially reducing immunogenicity to the mRNA.

[0521] Example 7

[0522] Effect of different UTR combinations on the expression of engineered meganucleases delivered by LNP in mice

[0523] 1. Methods and Materials

[0524] In these studies, protein levels of an engineered meganuclease (designated TTR 15-16x.81) that targets a recognition sequence in the mouse TTR gene (designated the TTR 15-16 recognition sequence) were measured in mouse liver using an antibody specific for the engineered meganuclease and a recombinant meganuclease protein standard in a sandwich ELISA on the MSD platform. The TTR 15-16x.81 meganuclease is described in PCT International Patent Application No. 2022 / 040528.

[0525] Mice were injected via the tail vein with either PBS alone or PBS plus LNPs at a dose of 2 mg of mRNA / kg body weight. LNPs contained TTR15-16x.81 Max mRNA (comprising the 5'XBG UTR of SEQ ID NO:7, the 3'XBG UTR of SEQ ID NO:12, N- and C-terminal c-myc NLSs, with the TTR15-16x.81 coding sequence codon-optimized for uridine deletion) (SEQ ID NO:188) or TTR15-16.81 Std mRNA (utilizing the standard control combination of the 5'HBA2 UTR, N-terminal SV40 sequence, and 3'HBA2 UTR) (SEQ ID NO:189). Three hours after injection, mice were euthanized, and the median lobe of the liver was collected and flash-frozen on dry ice. Approximately 40-90 mg of each liver was weighed and homogenized in MSD Tris lysis buffer containing complete Mini protease inhibitors using a SPEX MiniG 1600 tissue homogenizer. The total protein concentration of each lysate was determined by BCA, and the lysate was diluted to 1 mg / mL with MSD diluent 100. One MSD multi-array standard 96-well plate was coated overnight at 4°C with anti-meganuclease V34 antibody in PBS at a concentration of 4 μg / mL. Standards were prepared using recombinant meganuclease protein diluted to concentrations of 0-10 μg / mL in 1 mg / mL lysate from mice treated with PBS alone. The plate was blocked with 5% MSD Blocker A for 1 hour with shaking, washed three times with MSD Tris wash buffer, and then incubated with the lysate and standards for 90 minutes. The plates were washed again three times and incubated with sulfo-tagged anti-meganuclease M1 diluted to 1 μg / mL in PBS for 1 hour with shaking. The plates were then washed and MSD GOLD Read Buffer A was added to the wells. The plates were read using an MSD Quickplex SQ 120 instrument and the data analyzed using MSD Discovery Workbench software.

[0526] Example 8

[0527] Effect of optimized mRNA on meganuclease activity in targeting T cell receptors in vitro

[0528] 1. Methods and Materials

[0529] This experiment was conducted to compare the efficiency of an optimized mRNA formulation with a standard mRNA formulation encoding a TRC1-2-specific meganuclease in primary human T cells delivered by electroporation. Meganucleases targeting the TRC1-2 recognition sequence in the T cell receptor alpha constant region (TRAC) are described in PCT International Patent Application No. 2019 / 200122. In this paired study, apheresis samples were collected from healthy, informed, compensated donors, and T cells were enriched using the CD3 Positive Selection Kit II according to the manufacturer's instructions (Stem Cell Technologies). T cells were activated using ImmunoCult T cell stimulator (anti-CD2 / CD3 / CD28-Stem Cell Technologies) in Xuri medium (Cytiva) supplemented with 5% fetal bovine serum and 10 ng / mL IL-2 (Gibco). Three days after stimulation, cells were harvested and electroporated with either a standard mRNA formulation or a novel, optimized formulation (MAX formulation) of the TRC1-2 L.2307 meganuclease, which recognizes and cleaves the TRC1-2 site. The standard formulation was delivered in a two-fold titration ranging from 3540 ng per 1e6 cells to 13.8 ng per 1e6 cells. The MAX formulation was delivered in a two-fold titration ranging from 4000 ng per 1e6 cells to 62.5 ng per 1e6 cells.

[0530] After electroporation, cells were cultured for 3–5 days in complete Xuri medium supplemented with 30 ng / mL recombinant human IL-2 (medium changes were performed every 2–3 days). After at least 3 days of culture, cells were counted and stained for CD3 with either an APC-conjugated anti-CD3 antibody (Biolegend) or an FITC-conjugated anti-CD3 antibody (BioLegend). Data were acquired on a Beckman-Coulter CytoFLEX flow cytometer.

[0531] The DNA sequences of the constructs utilized in these experiments are listed in Table 11 below.

[0532] [Table 11]

[0533] 2.Results

[0534] Successful targeting of the TRAC gene at the TCR1-2 recognition site results in the loss of CD3 expression, resulting in CD3 knockout (KO) cells. A table showing the knockout frequencies of various experimental conditions is provided below.

[0535] [Table 12]

[0536] Dose-response curves for CD3 knockout with various doses of TRC1-2L.2307 meganuclease are shown in Figure 8, along with the EC90 and EC50 doses for each titration curve. In these dose-response curves, standard mRNA encoding TRC1-2L.2307 meganuclease was compared with Max mRNA. These mRNAs were delivered at a double dose by electroporation. As shown, Max mRNA reduced the EC90 and EC50 doses of TRC1-2L.2307 meganuclease by at least half. For example, electroporation of 125 ng of Max mRNA encoding TRC1-2L.2307 meganuclease knocked out CD3 in 78.61% of T cells, compared to 78.45% achieved with 442 ng of standard mRNA encoding TRC1-2L.2307 meganuclease.

[0537] 3. Conclusion

[0538] The results of this experiment demonstrate that the optimized Max mRNA encoding the TRC1-2L.2307 meganuclease outperformed standard mRNA in a study measuring TRAC-edited T cell knockout frequency by CD3 knockout. These results demonstrate that the optimized Max mRNA encoding the engineered meganuclease performs in a superior manner in a head-to-head comparison with standard mRNA formulations, consistent with other examples described herein utilizing different targeted meganucleases.

[0539] Example 9

[0540] Editing HAO1-2 recognition sequences in human cell lines using improved mRNA encoding the engineered HOA1-2L.30 meganuclease

[0541] 1. Methods and Materials

[0542] These studies were conducted using an in vitro cell-based system to evaluate whether improved mRNA design increased the in vitro editing efficiency of an engineered meganuclease designed to bind and cleave a target sequence (i.e., the HAO1-2 recognition sequence) within exon 8 of the HAO1 gene by digital PCR using an indel detection assay. The engineered meganuclease used in these experiments was the HAO1-2L.30 S19 meganuclease, encoded by the mRNA of SEQ ID NOs: 173-178. The HAO1-2L.30 meganuclease is described in PCT International Patent Application No. 2020 / 132659.

[0543] These studies were performed using an in vitro cell-based system and evaluated the editing efficiency of different HAO 1-2 meganucleases by digital PCR using an indel detection assay.

[0544] In these experiments, mRNAs utilizing combinations of 5' and 3' UTRs, along with combinations of additional N- and C-terminal NLSs as part of the engineered meganuclease, were tested against mRNAs utilizing the 5' HBA2 UTR and 3' WPRE UTR with an N-terminal NLS. Each mRNA in the experiment contained an N1-methylpseudouridine and 7-methylguanosine cap. Each meganuclease-encoding mRNA was electroporated into Hep3B cells at a dose of 2 ng using a Lonza Amaxa 4D system.

[0545] The mRNAs tested in this experiment are listed in Table 13.

[0546] [Table 13]

[0547] Cells were harvested 2, 6, and 9 days after electroporation for gDNA formulations and assessed for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency was greater than 90%. gDNA was prepared using the Macherey Nagel NucleoSpin Blood QuickPure kit.

[0548] Digital droplet PCR (Digital PCR) was used to determine the frequency of targeted insertions and deletions (indels%) in the HAO 1-2 recognition sequence using primers P1, F1, and R1, and primers P2, F2, and R2, to generate a reference amplicon. Amplification was multiplexed in a 20 μL reaction containing 1× ddPCR probe supermix (without dUTP, BioRad), 250 nM of each probe, 900 nM of each primer, 5 U of HindIII-HF, and approximately 50 ng of cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). The cycling conditions for HAO 1-2 were as follows: Cycling conditions for HAO 23-24 were: 1 cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 95°C (1°C / s ramp) for 30 seconds, 62°C (1°C / s ramp) for 30 seconds, 72°C (0.2°C / s ramp) for 2 minutes, 1 cycle of 98°C for 10 minutes, 4°C hold. Cycling conditions for HAO 23-24 were: 1 cycle of 95°C (2°C / s ramp) for 10 minutes, 44 cycles of 95°C (1°C / s ramp) for 30 seconds, 62°C (1°C / s ramp) for 30 seconds, 72°C (0.2°C / s ramp) for 2 minutes, 1 cycle of 98°C for 10 minutes, 4°C hold.

[0549] Droplets were analyzed using a QX200 droplet reader (BioRad), and data were acquired and analyzed using QuantaSoft analysis software (BioRad). Indel frequencies were calculated by dividing the number of positive copies of the binding site probe by the number of positive copies of the reference probe and comparing the loss of FAM+ copies in nuclease-treated cells with mock-transfected cells.

[0550] Primer set

[0551] P1:42 HAO1 2 BHQ 1 probe: TTCCTCACCAATGTCTTGT FAM (SEQ ID NO: 150) F1: 28-HAO21-22 F2: CCACATAAGATTTGGCAAGCC (SEQ ID NO: 151) R1: 27-HAO21-22 R2: GGAAAAGAACGACACCCTTTG (SEQ ID NO: 152) P2:33 HAO23 / 24 P1:CCCGGCTAATTTGTATCA VIC (SEQ ID NO: 153) F2:29-HAO23-24 f1:GCTCACTTGATGTAAGCAACAG (SEQ ID NO: 154) R2: 32-HAO23-24 R2: ACACACCACCAACGTAAAAC (SEQ ID NO: 155)

[0552] 2.Results

[0553] In these studies, indels (insertions and deletions) were measured by ddPCR at 2 ng per 0.5 e6 Hep3B cells. The percentage of indels was significantly enhanced using improved mRNA constructs with alternative UTRs and dual SV40 NLSs. At a 2 ng dose, the HAO1-2L.30 control meganuclease resulted in approximately 17% indel formation at day 9, while the best-performing modified construct, designated 35137 HAO1-2L.30, resulted in approximately 63% indel formation at day 9 (Figure 9). Similarly, a 2 ng dose of 35138 HAO1-2L.30 meganuclease resulted in approximately 62% indel formation at day 9, while the modified construct encoding 35114 HAO1-2L.30 meganuclease resulted in approximately 41% indel formation (Figure 9).

[0554] 3. Conclusion

[0555] These studies demonstrate the ability of the HAO 1-2 meganuclease to generate indels at the HAO 1-2 recognition sequence in Hep3B cells. The experiments further demonstrate that modifications to the mRNA encoding the meganuclease can significantly impact indel formation, resulting in much greater indel formation at lower mRNA doses (2 ng). This has the advantage of lowering the amount of mRNA that needs to be delivered to target cells and potentially reducing immunogenicity of the mRNA. Additionally, these studies demonstrate the hierarchy of UTR and NLS combinations for indel generation. Modifications of the 35137 HAO 1-2L.30 meganuclease resulted in 63% indels, increasing meganuclease expression beyond the 35114 construct encoding the same HAO 1-2L.30 meganuclease (which showed 41% indels). Thus, the trend toward increased indel formation over the control remained consistent across all other modifications, but differences were observed between the two types of UTRs and NLSs tested, allowing for the tunability of mRNA-based meganuclease expression.

[0556] Example 10

[0557] Effect of different UTR combinations on the expression of engineered meganucleases delivered by LNP in mice

[0558] 1. Methods and Materials

[0559] In these studies, protein levels of the engineered meganucleases listed in Table 14 were measured in mouse liver using antibodies specific for the engineered meganucleases and engineered meganuclease protein standards in a sandwich ELISA on the MSD platform.

[0560] Mice were injected via the tail vein with either PBS alone or LNP containing optimized (Max) or standard mRNA at a dose of 2 mg of mRNA / kg body weight. A complete description of each meganuclease-encoding construct is listed in Table 14. HBV11-12L.1090 meganuclease is described in PCT International Patent Application No. 2021 / 113765. The coding sequence of Max mRNA was codon-optimized for uridine deletion. These Max constructs contain the 5' XBG UTR of SEQ ID NO:7, the 3' XBG UTR of SEQ ID NO:12, and an N- or C-terminal cMYC NLS. Three hours after injection, mice were euthanized, and the median lobe of the liver was collected and flash-frozen on dry ice. Approximately 40-90 mg of each liver was weighed and homogenized in MSD Tris lysis buffer containing Complete Mini protease inhibitors using a SPEX MiniG 1600 tissue homogenizer. The total protein concentration of each lysate was determined by BCA, and the lysate was diluted to 1 mg / mL in MSD Diluent 100. One MSD Multi-Array Standard 96-well plate was coated overnight at 4°C with anti-meganuclease V34 antibody in PBS at a concentration of 4 μg / mL. Standards were prepared using standard engineered meganuclease protein diluted to concentrations of 0–10 μg / mL in 1 mg / mL lysate from mice treated with PBS alone. The plate was blocked with 5% MSD Blocker A for 1 hour with shaking, washed three times with MSD Tris wash buffer, and then incubated with the lysate and standard for 90 minutes. The plate was washed three times again and incubated with sulfo-tagged anti-meganuclease M1 diluted to 1 μg / mL in PBS for 1 hour with shaking. The plate was then washed, and MSD GOLD Read Buffer A was added to the wells. Plates were read using an MSD Quickplex SQ 120 instrument and data analyzed using MSD Discovery Workbench software.

[0561] [Table 14]

[0562] 2.Results

[0563] Livers of mice injected with standard mRNA encoding the HAO1-2 L.30S19 meganuclease showed protein expression ranging from 0.64 to 0.99 μg / g of tissue after collection 3 hours post-injection, whereas livers of mice injected with the optimized Max mRNA showed protein expression ranging from 0.99 to 1.61 μg / g of tissue. Similarly, livers of mice injected with HBV11-12 1090 Std mRNA showed protein expression ranging from 0.15 to 0.48 μg / g of tissue after collection 3 hours post-injection, whereas livers of mice injected with Max mRNA showed protein expression ranging from 0.5 to 1.3 μg / g of tissue.

[0564] 3. Conclusion

[0565] This experiment demonstrated the ability of LNP-delivered mRNAs encoding various engineered meganucleases to produce meganuclease protein in vivo. Furthermore, for the HAO1-2 L.30S19 and HBV11-12 L.1090 nucleases, mRNAs containing the XBG / XBG UTR, Cmyc NLS, and uridine deletion sequence produced more protein than standard control mRNAs containing the HBA2 / WPRE UTR, SV40 NLS, and non-uridine deletion sequence.

[0566] Example 11

[0567] Effect of different UTR combinations on the expression of engineered meganucleases delivered by LNP in mice

[0568] 1. Methods and Materials

[0569] In these studies, engineered meganuclease protein was measured in mouse liver using an engineered meganuclease-specific antibody and engineered meganuclease protein standards in a sandwich ELISA on the MSD platform.

[0570] Mice were injected via the tail vein with either PBS alone or LNP containing optimized Max or Std mRNA encoding each meganuclease at a dose of 0.3 mg of mRNA per kg of body weight. A complete description of the constructs is shown in Table 15. The HAO 25-26 meganuclease is described in PCT International Patent Application No. 2022 / 150616, and the TTR 15-16x.81 meganuclease is described in PCT International Patent Application No. 2022 / 040582. The coding sequence of each Max mRNA was codon-optimized for uridine deletion. Three hours after injection, mice were euthanized, and the median lobe of the liver was collected and flash-frozen on dry ice. Approximately 40-90 mg of each liver was weighed and homogenized in MSD Tris lysis buffer containing Complete Mini protease inhibitors using a SPEX MiniG 1600 tissue homogenizer. The total protein concentration of each lysate was determined by BCA, and the lysate was diluted to 1 mg / mL in MSD Diluent 100. One MSD Multi-Array Standard 96-well plate was coated overnight at 4°C with anti-meganuclease V34 antibody in PBS at a concentration of 4 μg / mL. Standards were prepared using standard engineered meganuclease protein diluted to concentrations of 0–10 μg / mL in 1 mg / mL lysate from mice treated with PBS alone. The plate was blocked with 5% MSD Blocker A for 1 hour with shaking, washed three times with MSD Tris wash buffer, and then incubated with the lysate and standard for 90 minutes. The plate was washed three times again and incubated with sulfo-tagged anti-meganuclease M1 diluted to 1 μg / mL in PBS for 1 hour with shaking. The plate was then washed, and MSD GOLD Read Buffer A was added to the wells. Plates were read using an MSD Quickplex SQ 120 instrument and data analyzed using MSD Discovery Workbench software.

[0571] [Table 15]

[0572] 2.Results

[0573] Livers from mice injected with HAO25-26L.1128 STD mRNA showed meganuclease protein expression ranging from 0.31 to 0.37 ng / mg of total protein after collection 3 hours post-injection, whereas livers from mice injected with HAO 25-26L.1128 Max mRNA showed meganuclease protein expression ranging from 0.94 to 1.5 ng / mg of total protein. Similarly, livers from mice injected with HAO25-26L.1434 STD mRNA showed meganuclease protein expression ranging from 0.5 to 0.6 ng / mg of total protein, whereas livers from mice injected with HAO 25-26L.1434 Max mRNA showed meganuclease protein expression ranging from 0.7 to 1.2 ng / mg of total protein.

[0574] 3. Conclusion

[0575] This experiment demonstrated the ability of LNP-delivered mRNAs encoding engineered meganucleases to produce meganuclease protein in vivo. Furthermore, for the HAO25-26L.1128 and HAO25-26L.1434 meganucleases, mRNAs containing the ALB / SNRPB UTR, SV40 NLS, and uridine deletion sequence produced more protein than standard control mRNAs containing the HBA2 / WPRE UTR, SV40 NLS, and non-uridine deletion sequence.

[0576] SEQ ID NO: 1 AATTATTGGTTAAAGAAGTATATTAGTGCTAATTTCCCTCCGTTTGTCCTAGCTTTTCTCTTCTGTCAACCCCACACGCCTTTGCCACC SEQ ID NO: 2 AGGTTGGGAACTAGGAGTGGCAGCAATCCTTTCTTTCAGCTGGAGTGCTCCTCAGGAGCCAGCCCCACCCTTAGCCACC SEQ ID NO: 3 ATAAGAGACCACAAGCGACCCGCAGGGCCAGACGTTCTTCGCCGAGAGTCGTCGGGGTTTCCTGCTTCAACAGTGCTTGGACGGAACCCGGCGCTCGTTCCCCACCCCGGCCGGCCGCCCATAGCCAGCCCTCCGTCACCTCTTCACCGCACCCTCGGACTGCCCCAAGGCCCCCGCCGCCGCTCCAGCGCCGCGCAGCCACCGCCGCCGCCGCCGCCTGCCACC SEQ ID NO:4 GCTCTCTGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCGTCGCCAGCCGAGCCACATCGCTCAGCCACC SEQ ID NO:5 CATAAACCCTGGCGCGCTCGCGGGCCGGCACTCTTCTGGTCCCCACAGACTCAGAGAGAAGCCA SEQ ID NO:6 GCATTTCCGGTAGCGGCGGCGGGAAATCGGCTGTGGGAGAGAGGCTAGGCCTCTGAGGAGGCGAATCCGGCGGGTATCAGAGCCATCAGAACCGCCAC SEQ ID NO:7 AAGCTCAGAATAAACGCTCAACTTTGGCC SEQ ID NO:8 GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAG SEQ ID NO:9 GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC SEQ ID NO: 10 ACTCATCTTGGCCCTCCTCAGCTCCCTGCCTGTTTCCCGTAAGGCTGTACATAGTCCTTTTATCTCCTTGTGGCCTATGAAACTGGTTTATAATAAACTCTTAAGAGAACATTA SEQ ID NO: 11 CCCTTGGCCACAGAGTATGGAAGTAGCTCCGCAGAGGCGTGGGCTCGATTCCTCAGGGCCACGTTACCACAGACCTGTTTGTTTCTTATGCTGTTGTTCGTGGAGTCTCATGGGATTGTCTGGTTTCCCTTACAGGGCCCCCTCCCCCGGGAATGCGCCCACCAAGGCCCTAGACTCATCTTGGCCCTCCTCAGCTCCCTGCCTGTTTCCCGTAAGGCTGTACATAGTCCTTTTATCTCCTTGTGGCCTATGAAACTGGTTTATAATAAACTCTTAAGAGAACATTA SEQ ID NO: 12 ACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCT SEQ ID NO: 13 ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCAGGGCTGCTCGCCTGTGTTGCCAACTGGATCCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCTCTCAATCCAGCGGACCTCCCTTCCCGAGGCCTTCTGCCGGTTCTGCGGCCTCTCCCGCGTCTTCGCTTTCGGCCTCCGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG SEQ ID NO: 14 GACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGCCGCATCTAGAGGGCC SEQ ID NO: 15 PKKKRKV SEQ ID NO: 16 RAAKRPRTT SEQ ID NO: 17 PAAKRVKLD SEQ ID NO: 18 HHPKKKRKV SEQ ID NO: 19 CCCAAGAAGAGCGCAAGGTG SEQ ID NO: 20 CGGGCCGCCAAGCGGCCACGGACCACC SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO:50 ACAAAA SEQ ID NO:51 ACAAAC SEQ ID NO:52 ACAAAG SEQ ID NO:53 AACCA SEQ ID NO:54 ACAACC SEQ ID NO: 55 ACAACG SEQ ID NO:56 ACAGAA SEQ ID NO:57 ACACAC SEQ ID NO:58 ACAGAG SEQ ID NO:59 ACAGCA SEQ ID NO: 60 ACACCC SEQ ID NO: 61 ACAGCG SEQ ID NO: 62 ACAAAA SEQ ID NO: 63 ACCAAC SEQ ID NO: 64 ACCAAG SEQ ID NO: 65 ACCACA SEQ ID NO: 66 ACCACC SEQ ID NO: 67 ACCACG SEQ ID NO: 68 ACCGAA SEQ ID NO: 69 ACCGAC SEQ ID NO: 70 ACCGAG SEQ ID NO: 71 ACCGCA SEQ ID NO:72 ACCGCC SEQ ID NO: 73 ACCCGCG SEQ ID NO:74 ATAAAA SEQ ID NO: 75 ATAAAC SEQ ID NO:76 ATTACK SEQ ID NO:77 ATAACA SEQ ID NO:78 ATAACC SEQ ID NO:79 ATTACK SEQ ID NO: 80 INTELLECTUAL SEQ ID NO: 81 ATAGAC SEQ ID NO:82 ATTITUDE SEQ ID NO: 83 ATTACK SEQ ID NO:84 ATAGCC SEQ ID NO: 85 ATAGCG SEQ ID NO:86 ATCAAA SEQ ID NO:87 ATCAAC SEQ ID NO: 88 ATCAAG SEQ ID NO:89 ATCACA SEQ ID NO: 90 ATCACC SEQ ID NO: 91 ATCACG SEQ ID NO:92 ATCGAA SEQ ID NO: 93 ATCGAC SEQ ID NO:94 ATCGAG SEQ ID NO: 95 ATCGCA SEQ ID NO:96 ATCGCC SEQ ID NO:97 ATCGCG SEQ ID NO: 98 GCAAAA SEQ ID NO: 99 GCAAAC SEQ ID NO: 100 GCAAAG SEQ ID NO: 101 GCAACA SEQ ID NO: 102 GCAACC SEQ ID NO: 103 GCAACG SEQ ID NO: 104 GCAGAA SEQ ID NO: 105 GCAGAC SEQ ID NO: 106 GCAGAG SEQ ID NO: 107 GCAGCA SEQ ID NO: 108 GCAGCC SEQ ID NO: 109 GCAGCG SEQ ID NO: 110 GCCAAA SEQ ID NO: 111 GCCAAC SEQ ID NO: 112 GCCAAG SEQ ID NO: 113 GCCACA SEQ ID NO: 114 GCCACC SEQ ID NO: 115 GCCACG SEQ ID NO: 116 GCCGAA SEQ ID NO: 117 CCCGAC SEQ ID NO: 118 GCCGAG SEQ ID NO: 119 GCCGCA SEQ ID NO: 120 GCCGCC SEQ ID NO: 121 GCCGCG SEQ ID NO: 122 GTAAAA SEQ ID NO: 123 GTAAAC SEQ ID NO: 124 GTAAAG SEQ ID NO: 125 GTAACA SEQ ID NO: 126 GTAACC SEQ ID NO: 127 GTAACG SEQ ID NO: 128 GTAGAA SEQ ID NO: 129 GTAAC SEQ ID NO: 130 GTAGAG SEQ ID NO: 131 GTAGC SEQ ID NO: 132 GTACC SEQ ID NO: 133 GTAGC SEQ ID NO: 134 GTCAAA SEQ ID NO: 135 GTCAAC SEQ ID NO: 136 GTCAAG SEQ ID NO: 137 GTCACA SEQ ID NO: 138 GTCACC SEQ ID NO: 139 GTCACG SEQ ID NO: 140 GTCGAA SEQ ID NO: 141 GTCGAC SEQ ID NO: 142 GTCGAG SEQ ID NO: 143 GTCGCA SEQ ID NO: 144 GTCGCC SEQ ID NO: 145 GTCGCG SEQ ID NO: 146 GGCACC SEQ ID NO: 147 GACACC SEQ ID NO: 148 CCCACC SEQ ID NO: 149 GGCCCC SEQ ID NO: 150 TTCCTCACCAATGTCTTGT SEQ ID NO: 151 CCACATAAGATTTGGCAAGCC SEQ ID NO: 152 GGAAAAGAACGACACCCTTTG SEQ ID NO: 153 CCCGGCTAATTTGTATCA SEQ ID NO: 154 GCTCACTTGATGTAAGCAACAG SEQ ID NO: 155 ACACACCACCAACGTAAAAC SEQ ID NO: 156 CCTCCCAGGAGTACTTCTCCAGG SEQ ID NO: 157 GATGCCTTCAGTGTCCTT SEQ ID NO: 158 CTTTGCTGACGTCCTAGT SEQ ID NO: 159 TACACGGGACACCTCACACCTG SEQ ID NO: 160 TGTGGTCACCCTCTGCACAGTGT SEQ ID NO: 161 TTGGATACAGCTTCCATCTA SEQ ID NO: 162 ACCAAACAAACAGTAAAATTGCC SEQ ID NO: 163 GAGGTCGATAAACGTTAGCCTC SEQ ID NO: 164 TGTGGTCACCCTCTGCACAGTGT SEQ ID NO: 165 CCACATAAGATTTGGCAAGCC SEQ ID NO: 166 TGTGGTCACCCTCTGCACAGTGT SEQ ID NO: 167 MAPKKKRKVH SEQ ID NO: 168 ATGGCCCCCAAGAAGAAGCGCAAGGTGCAT SEQ ID NO: 169 MNTKYNKEFLLYLAGFVDGDSIIAQIKPNQSYKFKHQLSLAFQVTQKTQRRWFLDKLVDEIGVGYVRDRGSVSDYILSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIWRLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP SEQ ID NO: 170 MNTKYNKEFLLYLAGFVDGDSIIAQIKPNQSYKFKHQLSLAFQVTQKTQRRWFLDKLVDEIGVGYVRDRGSVSDYILSEIKPLHNFLTQLQPFLKLKQKQQANLVLKIIEQLPSAKESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSAASSASSSPGSGISEALRAGAGSGTGYNKEFLYLAGFVDGDSIIAQIKPNQSYKFKHQLSLAFQVTQKTQRT SEQ ID NO: 171 SEQ ID NO: 172 SEQ ID NO: 173 SEQ ID NO: 174 SEQ ID NO: 175 SEQ ID NO: 176 SEQ ID NO: 177 SEQ ID NO: 178 SEQ ID NO: 179 SEQ ID NO: 180 SEQ ID NO: 181 SEQ ID NO: 182 SEQ ID NO: 183 SEQ ID NO: 184 SEQ ID NO: 185 SEQ ID NO: 186 SEQ ID NO: 187 SEQ ID NO: 188 SEQ ID NO: 189

Claims

1. A polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease, said nucleic acid sequence comprising: (a) a 5' untranslated region (UTR) comprising the nucleic acid sequence set forth in SEQ ID NO: 1; (b) a coding sequence encoding said engineered meganuclease; (c) a 3'UTR comprising the nucleic acid sequence set forth in SEQ ID NO: 10; (d) a polyA sequence, and (e) a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease; A polynucleotide wherein the engineered meganuclease comprises a first nuclear localization sequence (NLS) located at the N-terminus and a second NLS located at the C-terminus, and the first NLS and second NLS each comprise the amino acid sequence set forth in SEQ ID NO:

15.

2. the polynucleotide further comprises a modification to the coding sequence encoding the engineered meganuclease, whereby the thymidine or uridine content of the coding sequence is reduced; The polynucleotide of claim 1 , wherein the modification does not change the amino acid sequence of the engineered meganuclease.

3. The polynucleotide described in claim 1, wherein the promoter is a T7 promoter.

4. A polynucleotide described in claim 3, wherein the T7 promoter is a T7AG promoter.

5. A messenger RNA (mRNA) encoded by the polynucleotide described in claim 1.

6. The mRNA of claim 5 , wherein the mRNA comprises a 5′ cap.

7. The mRNA of claim 6, wherein the 5' cap comprises a 5' methylguanosine cap.

8. The mRNA according to claim 5, wherein the uridine present in the mRNA is pseudouridine or 2-thiouridine.

9. The mRNA of claim 5, wherein the uridines present in the mRNA are methylated.

10. The mRNA according to claim 9, wherein the uridine present in the mRNA is N1-methylpseudouridine, 5-methyluridine, or 2'-O-methyluridine.

11. A recombinant DNA construct comprising the polynucleotide of any one of claims 1 to 4.

12. A recombinant virus comprising the polynucleotide of any one of claims 1 to 4.

13. The recombinant virus of claim 12, wherein the recombinant virus is a recombinant adeno-associated virus (AAV).

14. A lipid nanoparticle composition comprising lipid nanoparticles containing the mRNA of any one of claims 5 to 10.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the lipid nanoparticle composition of claim 14.

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