5' untranslated regions (UTRS) for enhanced payload expression

Abstract

Described herein are polynucleotide compositions comprising 5' untranslated region (UTR) sequences that enhance translation of a payload under translational control of the 5' UTR. These 5' UTRs can enhance translation in various cell types, with some showing particular efficacy in specific cell types such as retinal pigment epithelium (RPE) cells relative to non-RPE cells. Also described herein are methods of expressing a payload in various target tissues, including but not limited to RPE, using a polynucleotide composition comprising the 5' UTR. The invention further provides libraries of 5' UTR sequences, methods for screening these libraries in multiple cell types, and recombinant polynucleotides incorporating the 5' UTRs for enhanced payload expression in various target cells, including RPE cells. These compositions and methods may be used for treating a wide range of disorders, with particular promise shown for retinal disorders through improved gene therapy approaches.

Description

KTS Ref.: 116779-1521686-740WO1Client Ref. No.: STX-130WO5’ UNTRANSLATED REGIONS (UTRS) FOR ENHANCED PAYLOAD EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos 63 / 694,433, filed September 13, 2024, and 63 / 783,753, filed April 4, 2025. The disclosure of the prior applications is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety; Said XML copy, created on September 9, 2025, is named “116779- 1521686-740WOl_SL.xml’' and is 188 kilobytes in size.BACKGROUND

[0003] The 5’ untranslated region (UTR) of mRNA plays a crucial role in regulating expression, for e.g., translation efficiency and overall protein expression. Therefore, the optimal use of the 5’ UTRs could significantly improve the efficacy of gene therapies targeting retinal disorders.SUMMARY

[0004] In various aspects, the present disclosure provides a recombinant polynucleotide comprising a 5’ untranslated region (5’ UTR). In some embodiments, the present disclosure provides recombinant polynucleotides with a 5’ untranslated region (5’ UTR) having a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the present disclosure provides recombinant polynucleotides with a 5 ’ untranslated region (5‘ UTR) having a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the present disclosure provides recombinant polynucleotides with a 5' untranslated region (5’ UTR) having a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the sequence of the 5' UTR has at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the sequence of the 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the 5’ UTR is any one of SEQ ID NO: 1-9 and SEQ ID-1-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WONO: 12-24. In some embodiments, the sequence of the 5’ UTR is SEQ ID NO: 2. In some embodiments, the sequence of the 5’ UTR is SEQ ID NO: 21. In some embodiments, the recombinant polynucleotide further includes a pay load operably linked to the 5’ UTR. In some embodiments, the present disclosure provides recombinant polynucleotides with a first 5’ untranslated region (5’ UTR) having a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24.

[0005] In various aspects, the present disclosure provides a recombinant polynucleotide comprising a first 5’ untranslated region (5’ UTR) having a sequence with at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

[0006] In some aspects, the sequence of the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12- 20, and SEQ ID NO: 22-24. In some aspects, the sequence of the first 5’ UTR is any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24. In some aspects, the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some aspects, the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some aspects, the first 5‘ UTR has a sequence with at least 90% sequence identity’ to SEQ ID NO: 2. In some aspects, the first 5?UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21. In some aspects, the first 5’ UTR has a sequence of SEQ ID NO: 2. In some aspects, the first 5’ UTR has a sequence of SEQ ID NO: 21.

[0007] In some embodiments, the present disclosure provides recombinant polynucleotides with a first 5‘ untranslated region (5' UTR) having a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the present disclosure provides recombinant polynucleotides with a first 5’ untranslated region (5’ UTR) having a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the sequence of the first 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the sequence of the first 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the first 5’ UTR is any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the sequence of the first 5’ UTR is SEQ ID NO: 2. In some embodiments, the sequence of the first 5’ UTR is SEQ ID NO: 21. In some embodiments, the recombinant polynucleotide further includes a first payload operably linked to the first 5 ’ UTR. In some embodiments, the recombinant polynucleotide further comprises a second 5’ UTR having a sequence with at least 80% sequence identity to any-2-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOone of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the polynucleotides as described herein further comprise a second 5’ UTR having a sequence with at least 80% sequence identity to any one of SEQ ID NO: 21, SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 3- 9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24, wherein the first and second 5‘ UTRs are different. In some embodiments, the sequence of the second 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 21, SEQ ID NO: 2; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24. In some embodiments, the sequence of the second 5’ UTR is any one of SEQ ID NO: 21. SEQ ID NO: 2; SEQ ID NO: 1, SEQ ID NO: 3-9. SEQ ID NO: 12-20, and SEQ ID NO: 22-24. In some embodiments, the recombinant polynucleotide further comprises a second 5’ UTR having a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the sequence of the second 5’ UTR has at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide further comprises a second 5’ UTR having a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the second 5’ UTR has at least 80% sequence identity' to SEQ ID NO: 21. In some embodiments, the recombinant polynucleotide further comprises a second 5’ UTR having a sequence with at least 90% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the recombinant polynucleotide further comprises a second 5' UTR having a sequence with at least 90% sequence identity’ to SEQ ID NO: 2. In some embodiments, wherein the sequence of the second 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide further comprises a second 5’ UTR having a sequence with at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, wherein the sequence of the second 5:UTR has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the sequence of the second 5’ UTR is any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the sequence of the second 5' UTR is SEQ ID NO: 2. In some embodiments, the sequence of the second 5’ UTR is SEQ ID NO: 21. In some embodiments, the first and second 5?UTRs are different. In some embodiments, the first 5?UTR is SEQ ID NO: 2 or SEQ ID NO: 21. In some embodiments, the second 5’ UTR is SEQ ID NO: 2 or SEQ ID NO: 21. In some embodiments, the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2 and the second 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the first 5' UTR has a sequence with at least 90% sequence identity’ to SEQ ID NO: 2 and the second 5’ UTR has a sequence with at least 90% sequence identity’ to SEQ ID NO: 21. In some embodiments, the first 5’ UTR is any one of SEQ ID NO: 2, SEQ ID NO: 1, and SEQ ID NO: 3-9, and the second 5’ UTR is any one of SEQ ID NO: 21,-3-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOSEQ ID NO: 12-20 and SEQ ID NO: 22-24. In some embodiments, the first 5’ UTR has a sequence of SEQ ID NO: 2 and the second 5’ UTR has a sequence of SEQ ID NO: 21. In some embodiments, the first 5’ UTR is SEQ ID NO: 2 and the second 5’ UTR is SEQ ID NO: 21. In some embodiments, the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21 and the second 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the first 5’ UTR has a sequence with at least 90% sequence identity7to SEQ ID NO: 21 and the second 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the first 5’ UTR has a sequence of SEQ ID NO: 21 and the second 5?UTR has a sequence of SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide further includes a second payload operably linked to the second 5’ UTR. In some embodiments, the first payload and / or the second payload comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide. In some embodiments, the gene is MECP2, PRPH2, RHO, UBE3A, DYRK1A, MEF2C. NSD1, ATRX, RPS6KA3, TCF4, ZEB2, FOXG1, CDKL5, a partial piece of chromosome 2, SLC6A1, DMD, SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1 A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR, FLCN, FLCN, TP53, CRP, PTEN, IFT88, CLDN14. AGT, MET, MYH9, YWHAE, HAMP, EPO, MUCL BAP1, APOE, CYBA. GSTT1, IFNG. IGF1, IL2. ABCB1, SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB, NOX4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT, CYBA, CYBB, EGFR, HM0X1, LRP2, SERPINE1, PAX2, ABCB1, PPARA, PPARG, PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRASL CRB2, APRT, AXL, CCND1, CBR1, CPT1A. CYP1A1, CYP2B6, EDNL ERBB2, HMGCR, MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2, PTGS2, HLTF, S0D1, SOD2, SREBF2, HNF1B, TERT, TNFSF10, NDRG1, MBTPS2, WNT4, BCOR, INF2, ALG9, BICC1, TMEM67, IRX2, FREM1, ANKS6, FREM2, CD46, COL4A3, COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9. In some embodiments, the recombinant polynucleotide further includes a promoter operably coupled to the payload, or a first promoter operably coupled to the first payload and a second promoter operably coupled to the second payload. In some embodiments, the recombinant polynucleotides as described herein further comprise (i) a first promoter operably coupled to both the first and second payloads, or (ii) a first promoter operably coupled to the first payload and a second promoter operably- coupled to the second payload. In some embodiments, the promoter comprises a core promoter having a polynucleotide sequence of any one of SEQ ID NO: 49 - SEQ ID NO: 208. In some embodiments, the core promoter has a polynucleotide sequence of SEQ ID NO: 52. In some-4-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the core promoter has a polynucleotide sequence of SEQ ID NO: 53. In some embodiments, each of the first and / or second promoter comprises a core promoter having a polynucleotide sequence of any one of SEQ ID NO: 49 - SEQ ID NO: 208. In some embodiments, the first promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 52. In some embodiments, the first promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 53. In some embodiments, the second promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 52. In some embodiments, the second promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 53. In some embodiments, the recombinant polynucleotide, upon insertion into a cell, results in increased expression of the payload in the cell of from about 1.5-fold to about 9- fold, relative to an otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 2-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 3-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 4-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5 ’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 5-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 6- fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 7-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 8-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 9-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5 ' UTR. In some embodiments, the cell is a neural-5-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOcell, retinal cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell.

[0008] In some embodiments, the recombinant polynucleotides as described herein further comprise a WPRE. In some embodiments, the WPRE comprises at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 214.

[0009] In some embodiments, the recombinant polynucleotides as described herein further comprise a polyA sequence. In some embodiments, the polyA is a bovine growth hormone polyA sequence. In some embodiments, the polyA sequence comprises at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 215.

[0010] In various aspects, the present disclosure provides a recombinant polynucleotide comprising: (i) a 5’ untranslated region (5’ UTR); and (ii) a pay load operably linked to the 5’ UTR, wherein the recombinant polynucleotide, upon insertion into a retinal pigment epithelium (RPE) cell, results in increased expression of the payload in the RPE cell of at least 2-fold, relative to an otherwise comparable recombinant polynucleotide lacking the 5 ’ UTR.

[0011] In various aspects, the present disclosure provides a recombinant polynucleotide comprising: (i) a first 5’ untranslated region (5’ UTR), (ii) a first pay load operably linked to the first 5’ UTR, (iii) a second 5’ UTR, and (iv) a second pay load operably linked to the second 5’ UTR; and wherein the recombinant polynucleotide, upon insertion into a retinal pigment epithelium (RPE) cell, results in increased expression of the first payload and / or second payload in the RPE cell of at least 2-fold, relative to an otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

[0012] Also provided herein are recombinant polynucleotides comprising a 5’ untranslated region (5’ UTR); and a payload operably linked to the 5’ UTR. In some embodiments, the 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the 5‘ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR is any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the 5’ UTR is SEQ ID NO: 2. In some embodiments, the 5’ UTR is SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a first 5‘ untranslated region (5‘ UTR),-6-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOa first payload operably linked to the first 5’ UTR, a second 5’ UTR, and a second payload operably linked to the second 5’ UTR. In some embodiments, the first 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 1-9 and the second 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 12-24. In some embodiments, the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9 and the second 5?UTR has at least 90% sequence identity to any one of SEQ ID NO: 12-24. In some embodiments, the first 5’ UTR is any one of SEQ ID NO: 1-9 and the second 5’ UTR is any one of SEQ ID NO: 12-24. In some embodiments, the first 5’ UTR has at least 80% sequence identity to SEQ ID NO: 2 and the second 5’ UTR has at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the first 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2 and the second 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the first 5’ UTR is SEQ ID NO: 2 and the second 5’ UTR is SEQ ID NO: 21. In some embodiments, the first 5‘ UTR has at least 80% sequence identity to any one of SEQ ID NO: 12-24 and the second 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 1-9. In some embodiments, the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 12-24 and the second 5’ UTR has at least 90% sequence identity7to any one of SEQ ID NO: 1-9. In some embodiments, the first 5’ UTR has a sequence of any one of SEQ ID NO: 12-24 and the second 5’ UTR has a sequence of any one of SEQ ID NO: 1-9. In some embodiments, the first 5’ UTR has at least 80% sequence identity to SEQ ID NO: 21 and the second 5' UTR has at least 80% sequence identity7to SEQ ID NO: 2. In some embodiments, the first 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21 and the second 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the first 5?UTR has a sequence of SEQ ID NO: 21 and the second 5’ UTR has a sequence of SEQ ID NO: 2. In some embodiments, the recombinant polynucleotide, upon insertion into a retinal pigment epithelium (RPE) cell, results in increased expression of the first and / or second payload(s) in the RPE cell of at least 2-fold, relative to an otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

[0013] Also provided herein is an engineered viral vector comprising the recombinant polynucleotide disclosed herein. In some embodiments, the adeno-associated viral vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and / or AAV-DJ.

[0014] The present disclosure also provides pharmaceutical compositions comprising the recombinant polynucleotide, the engineered viral vector, and a pharmaceutically acceptable carrier.

[0015] Also provided herein is a method of expressing a payload in a cell. Such methods include introducing a recombinant polynucleotide that includes a 5’ untranslated region (5’ UTR); and a-7-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOpayload operably linked to the 5’ UTR. In some embodiments, the 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the 5?UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the 5’ UTR has at least 90% sequence identity' to SEQ ID NO: 2. In some embodiments, the 5’ UTR has at least 90% sequence identity' to SEQ ID NO: 21. In some embodiments, the 5’ UTR is any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the 5’ UTR is SEQ ID NO: 2. In some embodiments, the 5’ UTR is SEQ ID NO: 21. In some embodiments, the methods include introducing a recombinant polynucleotide that includes a first 5’ untranslated region (5’ UTR), a first payload operably linked to the first 5‘ UTR, a second 5’ UTR, and a second pay load operably linked to the second 5‘ UTR. In some embodiments, the first 5‘ UTR has at least 80% sequence identity' to any one of SEQ ID NO: 1-9 and the second 5’ UTR has at least 80% sequence identity’ to any one of SEQ ID NO: 12-24. In some embodiments, the first 5’ UTR has at least 90% sequence identity’ to any one of SEQ ID NO: 1-9 and the second 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 12-24. In some embodiments, the first 5’ UTR is any one of SEQ ID NO: 1-9 and the second 5?UTR is any one of SEQ ID NO: 12-24. In some embodiments, the first 5 ' UTR has at least 80% sequence identity to SEQ ID NO: 2 and the second 5’ UTR has at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the first 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2 and the second 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the first 5‘ UTR is SEQ ID NO: 2 and the second 5’ UTR is SEQ ID NO: 21. In some embodiments, the first 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 12-24 and the second 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 1-9. In some embodiments, the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 12-24 and the second 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9. In some embodiments, the first 5’ UTR has a sequence of any one of SEQ ID NO: 12-24 and the second 5’ UTR has a sequence of any one of SEQ ID NO: 1-9. In some embodiments, the first 5’ UTR has at least 80% sequence identity to SEQ ID NO: 21 and the second 5’ UTR has at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the first 5?UTR has at least 90% sequence identity’ to SEQ ID NO: 21 and the second 5’ UTR has at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the first 5’ UTR has a sequence of SEQ ID NO: 21 and the second 5’ UTR has a sequence of SEQ ID NO: 2. In some embodiments, the payload is expressed at a-8-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOlevel that is from about 1.5-fold to about 9-fold higher than a control level of payload expression from inserting an otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR into the cell. In some embodiments, the payload is expressed a level that is about 2-fold higher than the control level of payload expression. In some embodiments, the pay load is expressed at a level that is about 3-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 4-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 5-fold higher than the control level of payload expression. In some embodiments, the pay load is expressed at a level that is about 6-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 7-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 8-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 9-fold higher than the control level of payload expression. In some embodiments, the cell is a neural cell, retinal cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell. In some embodiments, the cell is a retinal cell. In some embodiments, the retinal cell is a retinal pigment epithelium (RPE) cell. In some embodiments wherein the recombinant polynucleotide includes a 5’ untranslated region (5’ UTR) and a payload operably linked to a payload, the 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments, the sequence of the 5‘ UTR is any one of SEQ ID NO: 1-9 and SEQ ID NO: 12-24. In some embodiments wherein the recombinant polynucleotide includes a first 5' untranslated region (5’ UTR) operably linked to a first payload and a second 5 ’ UTR operably linked to a second payload, the first 5 ’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9, and the second 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 12-24. In some embodiments, the first 5’ UTR is any one of SEQ ID NO: 1-9 and the second 5’ UTR is any one of SEQ ID NO: 12-24. In some embodiments, the pay load comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide. In some embodiments, the payload, the first payload, the second payload, or any combination thereof, comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide. In some embodiments, the gene is MECP2, PRPH2, RHO, UBE3A, DYRK1 A, MEF2C, NSD1, ATRX, RPS6KA3, TCF4, ZEB2, FOXG1, CDKL5, a partial piece of chromosome 2, SLC6A1, DMD, SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1A, PKD1, PKD2, PKHD1, ACE, ALB, VHL,-9-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOEPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR, FLCN, FLCN, TP53, CRP, PTEN, IFT88, CLDN14, AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC1, BAP1, APOE, CYBA, GSTT1, IFNG, IGF1, IL2, ABCB1, SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB. N0X4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT. CYBA, CYBB, EGFR, HM0X1, LRP2. SERPINE1, PAX2, ABCB1, PPARA, PPARG, PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRAS1, CRB2, APRT, AXL, CCND1, CBR1, CPT1A, CYP1A1, CYP2B6, EDN1, ERBB2, HMGCR, MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2, PTGS2, HLTF. S0D1, SOD2, SREBF2. HNF1B, TERT, TNFSF10, NDRG1, MBTPS2, WNT4. BCOR. INF2, ALG9, BICC1, TMEM67, IRX2, FREM1, ANKS6, FREM2, CD46, COL4A3, COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9.

[0016] In various aspects, the present disclosure provides a method of treating a disorder in a subject in need thereof, the method comprising administering to the subject the recombinant polynucleotide as described herein, the engineered viral vector as described herein, or the pharmaceutical composition as described herein, thereby treating the disorder in the subject.

[0017] Also provided herein is a method of treating a disorder in a subject in need thereof. Such methods include administering to the subj ect the recombinant polynucleotide, the engineered viral vector, or the pharmaceutical composition disclosed herein, wherein the administering is sufficient to treat the disorder in the subject. In some embodiments, the disorder is selected from the group consisting of age-related macular degeneration (AMD), Leber’s hereditary optic neuropathy, cone-rod dystrophy, Leber congenital amaurosis, Stargardt’s disease, diabetic retinopathy, retinal detachment, Best's disease, retinitis pigmentosa, choroideremia, and tapetoretinal degeneration.INCORPORATION BY REFERENCE

[0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that-10-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0020] FIG. 1 is a schematic diagram illustrating the workflow for the 5' UTR library screen, including lentiviral transduction, cell sorting, and NGS analysis.

[0021] FIG. 2 is a series of scatter plots showing the correlation between biological replicates and different sorting bins from the HEK293T 5’ UTR library screen.

[0022] FIG. 3 is a set of scatter plots demonstrating the enrichment and depletion patterns of negative control sequences in different GFP expression bins from the HEK293T screen.

[0023] FIG. 4 is a series of box plots comparing the enrichment and depletion patterns of candidate 5’ UTR sequences across different GFP expression bins in HEK293T cells.

[0024] FIG. 5 is a scatter plot showing the statistical analysis of 5’ UTR enrichment in the 95- 100% GFP expression bin compared to the unsorted population in HEK293T cells.

[0025] FIG. 6 is a scatter plot showing polyserial regression analysis of 5' UTR sequence candidates' abundance across sorted GFP expression bins in HEK293T cells.

[0026] FIG. 7 is a bar graph comparing 5’ UTR enrichment analysis results from library screens in ARPE19 and HEK293T cells, using DESeq2 and polyserial regression-based testing strategies.

[0027] FIG. 8 is a schematic diagram of the dual luciferase AAV reporter construct used for validating 5’ UTR candidates.

[0028] FIG. 9 is a set of bar graphs showing NLuc and CLuc activities of HEK 293T cells transduced with AAV viruses with candidate 5’ UTRs and a bar graph of the fold-change in normalized NLuc activity for various 5’ UTR sequences compared to ano UTR control.

[0029] FIG. 10 is a set of bar graphs showing the ratio NLuc to CLuc activities, as well as the normalized fold-change, for various 5’ UTR sequences in ARPE19 cells.

[0030] FIG. 11 is a set of bar graphs showing the ratio NLuc to CLuc activities as well as the normalized fold-change for various 5‘ UTR sequences in iRPE cells.

[0031] FIG. 12 is a set of bar graphs showing the fold-change in normalized NLuc activity in transfected ARPE19 cells for various 5’ UTR sequences compared to a no UTR control.

[0032] FIG. 13 is a set of bar graphs showing the fold-change in normalized NLuc activity in transfected HEK cells for various 5’ UTR sequences compared to a no UTR control.DETAILED DESCRIPTION

[0033] Described herein are 5’ untranslated region (UTR) sequences and compositions thereof. Described herein are polynucleotide compositions comprising 5’ untranslated region (UTR) sequences. Further described herein are recombinant polynucleotides comprising a 5’ UTR. In-11-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsome embodiments, the recombinant polynucleotide comprises a first 5’ UTR and a second 5’ UTR. In some embodiments, the recombinant polynucleotide comprises an expression cassette. In some embodiments, the expression cassette comprises a 5’ UTR. In further embodiments, the expression cassette comprises a first 5' UTR and a second 5" UTR. In some embodiments, the 5’ UTR region is operably linked to a payload. Such UTR sequence can enhance expression of a payload in various cell types. In some embodiments, such UTR sequence can enhance expression of a payload in retinal pigment epithelium (RPE) cells. The term “payload expression’" is intended to encompass both payload transcription and payload translation. A 5’ UTR of the present disclosure may enhance pay load translation broadly across different cell types. When included upstream of a coding sequence, a 5’ UTR of the present disclosure may promote translation of a payload. A recombinant polynucleotide comprising a 5’ UTR of the present disclosure may have increased translation efficiency, which may be observed across various cell types, or in some cases, increased RPE specificity, or combinations thereof, compared to a recombinant polynucleotide with no 5’ UTR or with other 5’ UTRs. In embodiments where increased RPE specificity is observed, the 5’ UTR may increase efficacy of a payload (e.g., a therapeutic payload) in the RPE cells or tissues comprising RPE cells while reducing side effects resulting from off-target expression of the payload in non-RPE cells or tissues thereof. Additionally, in embodiments where translation enhancement is not cell-type specific, the 5’ UTRs may provide a general boost in protein expression across various cell ty pes, which can be beneficial for applications requiring increased payload expression in multiple tissue types.

[0034] A 5’ UTR of the present disclosure may be part of a polynucleotide construct to promote enhanced translation of the payload, which may be broadly applicable or, in some cases, specific to certain tissue or cell ty pes. A construct may7comprise a 5’ UTR, a coding sequence, and other regulatory7elements, and may regulate translation of a corresponding pay load. A construct may comprise a 5’ UTR, a coding sequence, and other regulatory7elements and promoters, and may regulate translation of a corresponding payload. Described herein are polynucleotide compositions comprising a 5’ UTR that can enhance payload translation in various contexts, including tissue type-specific or cell type-specific translation of the payload. Further described herein are polynucleotide compositions comprising the pay load, operably linked to the 5’ UTR. The polynucleotide compositions may comprise a recombinant polynucleotide. The polynucleotide compositions of the present disclosure may result in enhanced translation of the payload, which in some embodiments may be tissue type-specific or cell ty pe-specific. In some embodiments, the level of translation of the payload may depend on a cell type (e.g., RPE cells),-12-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOor tissue type (e.g., eye tissue), while in other embodiments, enhanced translation may be observed across multiple cell or tissue types.

[0035] A 5’ UTR as described herein may be selected for enhanced translation / expression of a payload described herein in a specific cell type (e.g., neuron, renal cell, hepatocyte, podocyte, retinal cell, epithelial cell, muscle cell, erythrocyte, platelet, bone marrow cell, vascular endothelial cell, lymphatic endothelial cell, epidermal cell, lymphocyte, myeloid cell, glial cell, interstitial cell, adipocyte, or fibroblast), a cell sub-type (e.g. venous vs. arterial vascular endothelium, excitatory' vs. inhibitory neurons, CD8 vs CD4 T cells), a cell state (e.g., diseased cell or healthy cell: activated or unactivated engineered cells, such as an activated or unactivated CAR T cell; myofibroblast or fibroblast; activated or resting T cell), a tissue type (e.g., diseased tissue, healthy tissue, nervous tissue (e.g., central nervous system, peripheral nervous system), kidney tissue, eye tissue, muscle tissue, blood, skin, fat, bone, cancerous tissue, thymus tissue, gastrointestinal tissue (e.g., stomach, intestine), spleen tissue, placenta tissue, pancreatic tissue, lung tissue, liver tissue, cardiac tissue, or brain tissue (e.g., cerebrum, cerebellum, adrenal)), or a combination thereof. In some embodiments, the enhanced translation of the payload occurs in neural cells, cells of the eye (including retinal cells, retinal pigment epithelium, and comeal cells), lung cells, epithelial cells, skeletal muscle cells, dendritic cells, hepatic cells, pancreatic cells, bone cells, hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, or heart cells. In some embodiments, the enhanced translation / expression of a payload occurs in retinal pigment epithelium (RPE) cells. In some embodiments, the enhanced translation / expression of a payload occurs in non-RPE cells. In some embodiments, a 5’ UTR can facilitate a higher amount of translation / expression of a payload in a specific cell type, relative to a second cell type. The 5’ UTR may be selected or engineered to tune the level of pay load translation broadly or in specific cell types. In some embodiments, the 5’ UTR may be selected or engineered to tune the level of pay load translation in the RPE (e.g., to a therapeutic level, such as about the same level as a wildtype version of a transgene in the RPE). In some embodiments, the 5’ UTR may be selected or engineered to tune the level of payload translation in non-RPE cells or tissue thereof (e.g., to below a level that produces off-target effects).Additionally, the 5’ UTR may be selected or engineered to enhance translation across multiple cell types when broader expression is desired. Tuning a translation level may comprise adjusting translation to a desired level. The translation level of the payload may control the expression level of the protein encoded by the pay load. For example, a high level of translation of a transgene may lead to a high level of expression of the protein encoded by the transgene.-13-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0036] Also described herein are methods of delivering a 5’ UTR to a subject. In some embodiments, the method of delivering is a method of delivering a recombinant polynucleotide comprising a 5’ UTR as described herein to a subject. In some embodiments, the method of delivering is a method of delivering a recombinant polynucleotide comprising a first 5' UTR and a second 5’ UTR as described herein to a subject. In some embodiments, the method of delivering is a method of delivering an expression cassette comprising a 5’ UTR as described herein to a subject. In some embodiments, the method of delivering is a method of delivering an expression cassette comprising a first 5" UTR and a second 5’ UTR as described herein to a subject. In some embodiments, the method of delivering is a method of delivering a polynucleotide composition of the present disclosure to a subject. In some embodiments, the method of delivering is a method of delivering a polynucleotide composition comprising a 5’ UTR of the present disclosure to a subject. In some embodiments, the method of delivering is a method of delivering a polynucleotide composition comprising a first 5’ UTR and a second 5?UTR of the present disclosure to a subject. In some embodiments, the polynucleotide composition may be encoded in a viral vector capable of delivering the polynucleotide to a cell of the subject. The viral vector may further comprise a viral capsid encapsidating the polynucleotide and facilitating delivery of the polynucleotide into the cell. A method of delivering a polynucleotide composition to a subject may comprise administering a viral vector comprising the polynucleotide to the subject. A method of delivering a polynucleotide composition to a subject may comprise administering a viral vector comprising the recombinant polynucleotide to the subject. Upon delivery of the polynucleotide to the subject, a pay load encoded by the polynucleotide may be translated in a cell of the subject with enhanced efficiency. A method of delivering a polynucleotide composition to a subject may comprise administering a viral vector comprising the expression cassette to the subject. Upon delivery of the recombinant polynucleotide to the subject, a payload encoded by the recombinant polynucleotide may be translated in a cell of the subject with enhanced efficiency. A method of delivering a polynucleotide composition to a subject may comprise administering a viral vector comprising the 5’ UTR to the subject. Upon delivery of the 5' UTR to the subject, a payload operably linked to the 5 ’ UTR may be translated in a cell of the subj ect with enhanced efficiency. A method of delivering a polynucleotide composition to a subject may comprise administering a viral vector comprising the first 5’ UTR and the second 5’ UTR to the subject. Upon delivery of the first 5’ UTR and the second 5’ UTR to the subject, a first payload operably linked to the first 5’ UTR may be translated in a cell of the subject with enhanced efficiency and a second pay load operably linked to the second 5‘ UTR may be translated in the cell of the-14-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsubject with enhanced efficiency. In some embodiments, this enhanced translation may occur in a cell type or tissue-type-dependent manner, while in other embodiments, enhanced translation may be observed across multiple cell or tissue types.

[0037] Further described herein are methods of treating a disease or condition by delivering a 5‘ UTR of the present disclosure to a subject and expressing a payload operably linked to the 5’ UTR in the subject with enhanced efficiency. Further described herein are methods of treating a disease or condition by delivering a first 5’ UTR and a second 5’ UTR of the present disclosure to a subject and expressing a first payload operably linked to the first 5’ UTR and a second pay load operably linked to the second 5’ UTR in the subject with enhanced efficiency. Further described herein are methods of treating a disease or condition by delivering a recombinant polynucleotide comprising a 5’ UTR of the present disclosure to a subject and expressing a pay load of the recombinant polynucleotide in the subject with enhanced efficiency. Further described herein are methods of treating a disease or condition by delivering a recombinant polynucleotide compnsing a first 5’ UTR and a second 5’ UTR of the present disclosure to a subject and expressing a first payload and a second payload of the recombinant polynucleotide in the subject with enhanced efficiency. Further described herein are methods of treating a disease or condition by delivering an expression cassette comprising a 5' UTR of the present disclosure to a subject and expressing a payload of the expression cassette in the subject with enhanced efficiency. Further described herein are methods of treating a disease or condition by delivering an expression cassette comprising a first 5’ UTR and second 5’ UTR of the present disclosure to a subject and expressing a first pay load and a second payload of the expression cassette in the subject with enhanced efficiency. Further described herein are methods of treating a disease or condition by delivering a polynucleotide composition of the present disclosure to a subject and expressing a pay load encoded by the polynucleotide in the subject with enhanced efficiency. In some embodiments, this enhanced expression may be cell type- or tissue typedependent, while in other embodiments, it may be broadly applicable across various cell or tissue types. The 5?UTR may be delivered as part of a viral vector. The first 5’ UTR and the second 5’ UTR may be delivered as part of a viral vector. The recombinant polynucleotide may be delivered as part of a viral vector. The expression cassette may be delivered as part of a viral vector. The polynucleotide composition may be delivered to the subject as part of a viral vector. The subject may have a disease or condition, for example a disease or condition caused by mutation or altered expression of a protein. In some embodiments, the 5’ UTR is operably linked to a transgene encoding a wildtype copy of the protein having the mutation or altered expression. In some embodiments, the first 5’ UTR and the second 5' UTR is operably linked to-15-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOa transgene encoding a wildtype copy of the protein having the mutation or altered expression. In some embodiments, the recombinant polynucleotide comprises a transgene encoding a wildty pe copy of the protein having the mutation or altered expression. In some embodiments, the expression cassette comprises a transgene encoding a wildtype copy of the protein having the mutation or altered expression. In some embodiments, the polynucleotide composition may comprise a transgene encoding a wildtype copy of the protein having the mutation or altered expression. The transgene may be translated with enhanced efficiency in a target cells or tissues of the subject upon delivery of the polynucleotide composition to the subject. In some embodiments, this enhanced translation may be selective to specific cell or tissue types, while in others it may be more broadly applicable. In some embodiments, a protein encoded by the transgene is expressed in the subject at a level dependent on the level of translation of the transgene. Translation of the transgene, expression of the protein encoded by the transgene, or both, with enhanced efficiency may treat the disease or condition in the subject. This enhanced efficiency may be cell type or tissue type-dependent in some cases, or may be more broadly applicable in others, depending on the specific therapeutic needs.

[0038] Also described herein are 5’ UTR libraries, methods of generating 5’ UTR libraries, and methods of screening 5’ UTR libraries to identify 5’ UTRs that promote enhanced translation, which may be broadly applicable or specific to certain tissue or cell types. A 5’ UTR library may comprise candidate 5’ UTRs expected to promote enhanced translation of nearby sequences, including those that may exhibit tissue type- or cell-type-specific effects, along with reference sequences for cross-comparison. The libraries may be screened using high-throughput screening to assess translation in various cellular models, including but not limited to the cell or tissue of interest (e.g., RPE or RPE cells, or RPE cell lines) or in animal models (e.g., mice, rats, dogs, non-human primates). This screening may evaluate both general translation enhancement and potential tissue- or cell-tj pe specificity.

[0039] Also described herein are recombinant polynucleotides for expressing a pay load (e g., a transgene) with enhanced efficiency. The recombinant polynucleotide for expression a payload (e.g., a transgene) with enhanced efficiency may comprise a 5’ UTR of the present disclosure. The recombinant polynucleotide for expression a first payload (e.g., a first transgene) and a second pay load (e.g., a second transgene) with enhanced efficiency may comprise a first 5’ UTR and a second 5’ UTR of the present disclosure. A recombinant polynucleotide of the present disclosure can be used to treat a disease or disorder caused by a mutation, deletion, or altered expression of a gene. For example, a recombinant polynucleotide can be used to treat a disease or disorder caused by a mutation, deletion, or altered expression of a gene by expressing a-16-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOwildt pe copy of the gene encoded by the recombinant polynucleotide. Various sequence elements, such as 5’ UTRs, introns, post-transcriptional regulatory elements, or poly adenylation signals can affect translational levels of a payload encoded by the recombinant polynucleotide. Described herein are sequence elements and recombinant polynucleotides comprising sequence elements for expression of a payload. These sequence elements may provide general translation enhancement or, in some cases, tissue- or cell-type specific effects. These sequence elements may provide general translation enhancement or, in some cases, tissue- or cell-type specific effects in addition to promoters. Variation of the sequence elements included in a recombinant polynucleotide can be used to tune expression levels of a payload encoded by the recombinant polynucleotide, allowing for optimization of expression across various cell ty pes or for specific target tissues.

[0040] A recombinant polynucleotide can comprise a 5’ UTR and a coding sequence. A recombinant polynucleotide can comprise a first 5’ UTR and a first coding sequence, and a second 5‘ UTR and a second coding sequence. In some embodiments, the recombinant polynucleotide includes additional sequence elements that enhance translation of the payload. In some embodiments, the recombinant polynucleotide includes additional sequence elements that enhance translation of the pay load in addition to promoters. This enhancement may be general or cell-type specific, depending on the specific elements used. Examples of additional sequence elements that can be included in a recombinant polynucleotide are an intron, a polyadenylation signal (polyA signal) encoding a signaling addition of a polyadenylation tail (polyA tail), a post- transcriptional regulatory element, or any combination thereof. For example, a recombinant polynucleotide includes a 5’ UTR. a coding sequence, a post-transcriptional regulatory element, and a polyadenylation signal. For example, a recombinant polynucleotide includes a promoter, a 5’ UTR, a coding sequence, a post-transcriptional regulatory element, and a polyadenylation signal. A coding sequence encoding the payload can be operably linked to a 5’ UTR sequence, such that the 5‘ UTR regulates transcription of the pay load. A coding sequence encoding the payload can be operably linked to a 5’ UTR sequence, such that the 5’ UTR regulates translation of the payload. A coding sequence encoding the payload can be operably linked to a 5’ UTR sequence and a promoter, such that the 5’ UTR regulates translation of the payload. For example, a recombinant polynucleotide includes a first 5' UTR, a first coding sequence, a post- transcriptional regulatory element, and a polyadenylation signal, and further comprises a second 5’ UTR, a second coding sequence, a post-transcriptional regulatory element, and a polyadenylation signal. The first coding sequence encoding a first payload can be operably linked to the first 5’ UTR, such that the first 5’ UTR regulates translation of the first payload,-17-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOand the second coding sequence encoding a second payload can be operably linked to the second 5’ UTR, such that the second 5’ UTR regulates translation of the second payload. As another example, a recombinant polynucleotide includes a first promoter, a first 5’ UTR, a first coding sequence, a post-transcriptional regulatory element, and a polyadenylation signal, and further comprises a second promoter, a second 5’ UTR. a second coding sequence, a post-transcriptional regulatory element, and a polyadenylation signal. The first coding sequence encoding a first payload can be operably linked to the first promoter and the first 5’ UTR, such that the first 5’ UTR regulates translation of the first pay load, and the second coding sequence encoding a second pay load can be operably linked to the second promoter and the second 5’ UTR. such that the second 5’ UTR regulates translation of the second pay load.

[0041] The recombinant polynucleotide can be used to treat a disease or disorder. In some embodiments, the recombinant polynucleotide can be delivered via viral vector to treat a disease or disorder to a subject in need thereofRecombinant Polynucleotides

[0042] A recombinant polynucleotide may comprise a 5’ UTR of the present disclosure. A recombinant polynucleotide may comprise a first 5’ UTR and a second 5’ UTR of the present disclosure. The first 5’ UTR may be different than the second 5’ UTR. The first 5’ UTR may be the same as the second 5?UTR. The first 5’ UTR may be any 5’ UTR of the present disclosure. The second 5’ UTR may be any 5’ UTR of the present disclosure. A recombinant polynucleotide may comprise a coding sequence. The coding sequence may be operably linked to a 5’ UTR of the present disclosure. The coding sequence may be a payload sequence for expression of the payload. A recombinant polynucleotide may comprise a first coding sequence and a second coding sequence. The first coding sequence may be operably linked to a first 5 ' UTR of the present disclosure and the second coding sequence may be operably linked to a second 5’ UTR of the present disclosure. A recombinant polynucleotide may comprise a payload sequence (e.g., a transgene encoding a protein) operably linked to a promoter. The payload sequence may be operably linked to a 5’ UTR of the present disclosure. A recombinant polynucleotide may comprise a payload sequence (e.g., a transgene encoding a protein) operably linked to a promoter and a 5’ UTR of the present disclosure. In some embodiments, the pay load sequence encodes a protein (e.g., a therapeutic protein). In some embodiments, the recombinant polynucleotide may comprise two payload sequences (e.g., two transgenes, each encoding a protein). For example, the recombinant polynucleotide may comprise a first payload sequence (e.g., a first transgene encoding a first protein) and a second payload sequence (e.g., a second transgene encoding a second protein). For example, the recombinant poly nucleotide may-18-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOcomprise a first payload sequence (e.g., a first transgene encoding a first protein) operably linked to a first 5’ UTR of the present disclosure and a second payload sequence (e.g., a second transgene encoding a second protein) operably linked to a second 5’ UTR of the present disclosure. As another example, the recombinant polynucleotide may comprise a first payload sequence (e.g.. a first transgene encoding a first protein) operably linked to a first promoter and a first 5’ UTR of the present disclosure and a second payload sequence (e.g., a second transgene encoding a second protein) operably linked to a second promoter and a second 5 ' UTR of the present disclosure. In some embodiments, each of the two pay load sequences encodes a protein (e.g., a therapeutic protein) that is different from the other. For example, a first pay load sequence is different from the second payload sequence. As another example, the first payload sequence and the second payload sequence encode different proteins. In some embodiments, the two payload sequences encode for the same protein. For example, the first payload sequence is the same as the second payload sequence or the first payload sequence and the second payload sequence encode for the same protein.

[0043] The recombinant polynucleotide can comprise a polynucleotide cassette coding for expression of a coding sequence, also referred to as an “expression cassette”. An expression cassette can comprise one or more coding sequences (e.g., one or more payload sequences) and one or more regulatory sequences (e.g., a promoter, a 5’ UTR, a 3’ UTR. a post-transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a CCCTC-binding factor sequence, a 5’ stuffer sequence, or combinations thereof) operably linked to the coding sequence to regulate transcription and / or translation of the coding sequence. The expression cassette can comprise a 5’ UTR of the present disclosure. The expression cassette can comprise a first 5’ UTR and a second 5’ UTR of the present disclosure. In cases where the expression cassette comprises more than one coding sequence (e.g., payload sequence), each coding sequence may be operably linked to its own set of regulatory7sequences. For example, an expression cassette can comprise a first 5‘ UTR operably linked to a first coding sequence and a second 5‘ UTR operably linked to a second coding sequence. These regulatory sequences may be selected or engineered to enhance expression broadly across various cell types or to provide cell-type specific regulation, depending on the desired application. The expression cassette can comprise a promoter operably linked to the coding sequence (e.g., the payload sequence) may comprise a core promoter and an enhancer. The expression cassette can comprise a promoter and a 5' UTR operably linked to a coding sequence (e.g., the payload sequence), wherein the promoter may comprise a core promoter and an enhancer. The core promoter and the enhancer may regulate expression of the payload sequence (and therefore the payload encoded by the-19-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOpayload sequence) in a manner that can be tailored to specific therapeutic needs. In some embodiments, the expression cassette comprise a first promoter operably linked to a first coding sequence (e.g., a first pay load sequence) and second promoter operably linked to a second coding sequence (e.g., a second payload sequence). In some embodiments, the expression cassette comprises a first promoter and a first 5’ UTR operably linked to a first coding sequence (e.g., a first payload sequence) and second promoter and a second 5’ UTR operably linked to a second coding sequence (e.g., a second payload sequence). The first promoter and the second promoter can be different. The first promoter and the second promoter can be the same. The first promoter can comprise a first core promoter and a first enhancer. The second promoter can comprise a second core promoter and a second enhancer. In some embodiments, the first core promoter and the second core promoter are the same. In some embodiments, the first core promoter and the second core promoter are different. In some embodiments, the first enhancer and the second enhancer are the same. In some embodiments, the first enhancer and the second enhancer are different. In some embodiments, the expression cassette comprises a first core promoter and a second core promoter that are different and a first enhancer and a second enhancer that are different. In some embodiments, the expression cassette comprises a first core promoter and a second core promoter that are the same and a first enhancer and a second enhancer that are different. In some embodiments, the expression cassette comprises a first core promoter and a second core promoter that are different and a first enhancer and a second enhancer that are the same. In some embodiments, the expression cassette comprises a first core promoter and a second core promoter that are the same and a first enhancer and a second enhancer that are the same.

[0044] In some embodiments, the recombinant polynucleotide, upon insertion into a cell, results in increased expression of the payload in the cell of from about 1.5-fold to about 9-fold, relative to an otherwise comparable recombinant polynucleotide lacking the 5’ UTR. In some embodiments, the recombinant polynucleotide, upon insertion into a cell, results in increased expression of two payloads in the cell of from about 1.5-fold to about 9-fold, relative to an otherwise comparable recombinant polynucleotide lacking the 5’ UTR for those payloads. For example, the recombinant polynucleotide comprises a first transgene for expression of first payload operably linked to a first 5’ UTR and a second transgene for expression of the second payload operably linked to a second 5’ UTR. When the recombinant polynucleotide comprises two payloads, the two payloads can have the same transgene sequence or they can have different transgene sequences. In many cases, each of the two payloads may be operably linked to a 5’ UTR sequence that is the same as or a 5’ UTR sequence that is different from the other-20-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO5’ UTR sequence. In many cases, expression levels of the two different payloads in the cell can be about the same when compared to each other or they can be different.

[0045] In some embodiments, the recombinant poly nucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 2-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 3-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 4-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5 ’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 5-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 6-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 7-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5 ’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 8-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5’ UTR(s)). In some embodiments, the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload(s) in the cell of from about 9-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR (e.g., the corresponding 5' UTR(s)).5’ UTR

[0046] A payload of the present disclosure may comprise a sequence operably linked to a 5’ untranslated region (UTR). The 5' UTR may be a 5’ UTR of the present disclosure. The payload may comprise a transgene for delivery to various cell types, including but not limited to a neural cell, retinal cell e.g., retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell, of a human or-21-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOnon-human subject. The 5’ UTR is designed to enhance translation efficiency of the downstream coding sequence, which may be observed broadly across different cell types or, in some cases, particularly in RPE cells. These 5’ UTRs can be utilized to achieve enhanced translation in a variety of cellular contexts, with some showing particular efficacy in specific cell types such as RPE. The 5’ UTRs may be selected from SEQ ID NO: 1-9 and SEQ ID NO: 12-24, as shown in TABLE 1TABLE 1 - 5’ UTRs-22-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO-23-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0047] Various control sequences may be used to test the efficacy of the 5’ UTR sequences disclosed herein, such as, but not limited to, TGGCGAAGAGGGGCGGAGTCACGAGCGGGGCGGTGAGACTTCCTGCCATGTCGCGG GCCAGCCTAGCGCTTCAGCCGGCGGCTCATTTCCGGTGGGGGATGCGCGCCCAGTGA GGGCCCGGAAGTGGGTCGCGCGGAGATTGCTGGGCATGTCTTGCCGGAAGCGGAGA GCGGCTGATCGCAGTCCGGAGGTGGTCCATT (SEQ ID NO: 10), and GAGGTAGGGACGTGCTGTAGGCCGGAGGGAGTGGCGTGCTGGGCGTGATGGGCTGC GGTACGGCGTGTTGGTCCCAGCGGTTCAGCTGAGGTAGGGACATGCTGTAGGCCGG AGGGAGTGGCGTGCTGGGCGTGCGCGGCTGCGGTACATGGTGTTGGTCCCAGCGGT TCAGCTGAGGTAGGGACGTGCTGTAGGCCGGA (SEQ ID NO: 11).

[0048] The 5’ UTR sequences of the present invention are selected from a library of candidate sequences designed to promote efficient translation initiation and enhance overall protein expression in various cell types. These 5’ UTRs may be derived from various sources, including synthetic 5‘ UTR designs based on computational models and previous optimization studies, endogenous 5’ UTRs identified through matched Ribo-Seq and RNA-Seq data from multiple cell Npes, such as neural cell, retinal cell e.g., retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte. fibroblast, endothelial cell, prostate cell, or heart cell, 5’ UTRs designed based on single-nucleus data from various cell types such as RPE cells and neurons, and combinations and variations of known translational enhancing elements.

[0049] The 5’ UTRs of the present invention may enhance translation efficiency in multiple cell types, with some showing particular efficacy in specific cell types such as RPE cells, through various mechanisms. These mechanisms may include improving ribosome recruitment and translation initiation, reducing secondary structure that may impede ribosome scanning, incorporating regulatory elements that enhance translation in different cellular environments, and optimizing codon usage for efficient translation in target cell types.

[0050] In some embodiments, a transgene comprising a 5?UTR of the present invention may be translated in target cell types, such as neural cell, retinal cell e.g., retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell, at a level that is at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4- fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least-24-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOabout 8.5-fold, at least about 9-fold, or greater than 9-fold higher than the translation level of the same coding sequence lacking the 5 ’ UTR.

[0051] The 5’ UTRs may be particularly effective when combined with cell-type specific or broadly active promoters or enhancers to achieve both transcriptional and translational enhancement of payload expression in target cells. This combination may allow for fine-tuning of gene expression levels in the desired cell types.

[0052] In some embodiments, the payload may comprise a therapeutic protein-coding sequence. The enhanced translation efficiency provided by the 5’ UTRs may allow for lower doses of the therapeutic transgene to achieve desired protein expression levels, potentially reducing off-target effects or vector-related toxicity. This may be particularly beneficial for gene therapy applications targeting various disorders, including retinal disorders.

[0053] The 5’ UTRs of the present invention may be used in conjunction with various types of transgenes across multiple cell and tissue types. These may include wild-type copies of genes mutated in various diseases, including but not limited to retinal diseases, therapeutic proteins for treating or preventing a wide range of disorders, with particular efficacy shown in retinal disorders, reporter genes for assessing payload expression and distribution in different cellular contexts, and genes encoding regulatory RNAs such as shRNAs or guide RNAs for gene editing applications.

[0054] The effectiveness of the 5’ UTRs may be assessed using reporter systems, such as fluorescent proteins or luciferases, allowing for quantitative measurement of translational enhancement in both in vitro and in vivo models across various cell types and tissues. These reporter systems may provide valuable data on the performance of different 5‘ UTR sequences (SEQ ID NO: 1-9 and 12-24) in various cellular contexts and help guide the selection of optimal sequences for specific therapeutic applications in a wide range of tissues and disorders.

[0055] In some embodiments, the 5‘ UTR may comprise a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%. at least about 85%. at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the 5’ UTR may comprise a sequence having at least about 70%, at least about 72%. at least about 75%. at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the-25-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO5’ UTR may comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%. or about 100% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%. at least about 95%, at least about 97%, at least about 98%, at least about 99%. or about 100% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the 5‘ UTR may comprise a sequence having at least about 96% sequence identity to SEQ ID NO: 2. In some embodiments, the 5' UTR may comprise a sequence having at least about 97% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the 5‘ UTR may comprise a sequence of SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 96% sequence identity’ to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 97% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 21. In some embodiments, the 5‘ UTR may comprise a sequence of SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 96% sequence identity’ to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 97% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 6. In some embodiments, the 5‘ UTR may comprise a sequence having at least about 99%-26-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the 5’ UTR may comprise a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity7to SEQ ID NO: 21. In some embodiments, the 5' UTR may comprise a sequence having at least 70%. at least 72%. at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the 5‘ UTR may comprise a sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 96% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 97% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the 5' UTR may comprise a sequence having at least 95% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least 96% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least 97% sequence identity7to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least 98% sequence identity to SEQ ID NO: 21. In some embodiments, the 5?UTR may comprise a sequence having at least 99% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may-27-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOcomprise a sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least 96% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least 97% sequence identity to SEQ ID NO: 6. In some embodiments, the 5‘ UTR may comprise a sequence having at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 70%, about 72%, about 75%, about 78%. about 80%, about 82%, about 85%, about 87%. about 90%. about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the 5’ UTR may comprise a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%. about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%. about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having about 70%, about 72%. about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity7to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 2. In some embodiments, the 5‘ UTR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 100% of SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having about 95% sequence identity’ to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having about 96% sequence identity’ to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 21. In some-28-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the 5’ UTR may comprise a sequence having about 98% sequence identify to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having about 99% sequence identify to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having about 100% sequence identify to SEQ ID NO: 21. In some embodiments, the 5?UTR may comprise a sequence having about 90% sequence identify to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 95% sequence identify to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 96% sequence identify to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 97% sequence identify to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 98% sequence identify to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 99% sequence identify to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having about 100% of SEQ ID NO: 6.

[0056] In some embodiments, the 5’ UTR may comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identify’ to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the 5’ UTR may comprise a sequence having 70%. 72%. 75%, 78%, 80%, 82%, 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identify to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identify to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 70%, 72%. 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identify' to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 90% sequence identify to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 95% sequence identify to SEQ ID NO: 2. In some embodiments, the 5' UTR may comprise a sequence having 96% sequence identify to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 97% sequence identify to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 98% sequence identify to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 99% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 2. In some embodiments, the 5?UTR may comprise a sequence having 90% sequence identify to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 95% sequence identify to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 96% sequence identify to SEQ ID-29-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WONO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 97% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 98% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 99% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 90% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 95% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 96% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 97% sequence identity’ to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 98% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 99% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 6.

[0057] In some embodiments, the recombinant polynucleotide comprises one 5 ’ UTR. In some embodiments, the recombinant polynucleotide comprises two 5’ UTRs (e.g., a first 5’ UTR and a second 5' UTR) and the two 5' UTRs have the same sequence. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) each comprise a sequence having at least about 70%, at least about 72%. at least about 75%. at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%. or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 80% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5‘ UTR) may each comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5' UTRs (e g., the first 5' UTR and the second 5‘ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 80% sequence identity to SEQ-30-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 95% sequence identity’ to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having about 80% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5' UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 98% sequence identity7to SEQ ID NO: 2. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 100% sequence identity to SEQ ID NO: 2.

[0058] In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 80% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 90% sequence identity^ to SEQ ID NO: 2. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5‘ UTR and the second-31-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO5’ UTR) may each comprise a sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 95% sequence identity' to SEQ ID NO: 2. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5' UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having about 80% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5‘ UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 80% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 99% sequence identity7to SEQ ID NO: 21. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise SEQ ID NO: 21. In some embodiments, the two 5?UTRs (e.g., the first 5?UTR and the second 5’ UTR) may each comprise a sequence having at least 80% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 90% sequence identity to SEQ ID NO:-32-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 95% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5' UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having at least 98% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having at least 99% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having about 80% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having about 90% sequence identity to SEQ ID NO: 21 . In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5' UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having about 98% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 99% sequence identity7to SEQ ID NO: 21. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having about 100% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 80% sequence identity7to SEQ ID NO: 6. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may-33-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOeach comprise a sequence having at least 95% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least 98% sequence identity7to SEQ ID NO: 6. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 80% sequence identity7to SEQ ID NO: 6. In some embodiments, the two 5' UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having about 90% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having at least about 95% sequence identity7to SEQ ID NO: 6. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having about 98% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having about 99% sequence identity7to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having about 100% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity7to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5‘ UTR) may each comprise a sequence having 80% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 90% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each comprise a sequence having 95% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g.. the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 98% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5 ’ UTR and the second 5 ’ UTR) may each comprise a sequence having 99% sequence identity to SEQ ID NO: 2. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 80% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 90% sequence identity7to SEQ ID NO: 21. In some embodiments, the two 5‘ UTRs (e.g., the first 5‘ UTR and the second 5’ UTR) may each-34-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOcomprise a sequence having 95% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 98% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5 ’ UTR and the second 5 ' UTR) may each comprise a sequence having 99% sequence identity to SEQ ID NO: 21. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 80% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 90% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5?UTR) may each comprise a sequence having 95% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) may each comprise a sequence having 98% sequence identity to SEQ ID NO: 6. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5' UTR) may each comprise a sequence having 99% sequence identity to SEQ ID NO: 6.

[0059] In some embodiments, the recombinant polynucleotide comprises two different 5’ UTRs. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%. at least about 80%, at least about 82%, at least about 85%. at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24: (2) the second 5‘ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; and the first and the second 5’ UTRs have different sequences (e.g., different SEQ ID NOs). In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24: (2) the second 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least about 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID-35-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WONO: 24; and the first and the second 5’ UTRs have different sequences (e.g., different SEQ ID NOs). In some embodiments, (1) the first 5’ UTR comprises a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%. about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity- to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; (2) the second 5’ UTR comprises a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%. about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; and the first and the second 5’ UTRs have different sequences (e.g., different SEQ ID NOs).

[0060] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity7to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; and (3) and the first and the second 5’ UTRs have different sequences (e.g., different SEQ ID NOs).

[0061] In some embodiments, (1) the first 5?UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9; and (2) the second 5:UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%. at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9; and (2) the second 5?UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, (1) the-36-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOfirst 5’ UTR comprises a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9; and (2) the second 5’ UTR comprises a sequence having about 70%, about 72%, about 75%. about 78%, about 80%, about 82%, about 85%. about 87%.at least about 90%. about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24.

[0062] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9; and (2) the second 5' UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24.

[0063] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having at least about 70%. at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%. at least about 99%, or about 100% sequence identity' to any one of SEQ ID NO: 12- SEQ ID NO: 24.

[0064] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24.

[0065] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%. at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%. at least about 97%. at least about 98%, at least about 99%, or about 100% sequence identity7to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about-37-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21.

[0066] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%. at least 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity7to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 70%, about 72%, about 75%. about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21.

[0067] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 70%, at least 72%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%. at least 99%. or 100% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having about 70%, about 72%, about 75%. about 78%, about 80%, about 82%, about 85%, about 87%, about 90%, about 92%, about 93%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%,-38-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6.

[0068] In some embodiments. (1) the first 5?UTR comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 90% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having at least about 90% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 95% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 96% sequence identity' to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 97% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 97% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 98% sequence identity’ to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 98% sequence identity' to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 99% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 99% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 80% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 80% sequence identity’ to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 95% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 95% sequence identity' to SEQ ID NO: 21. In some embodiments, (1) the first 5‘ UTR comprises a sequence having at least 96% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having at least 96% sequence identity’ to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 97% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 97%-39-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 98% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5‘ UTR comprises a sequence having at least 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 99% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 80% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 80% sequence identity' to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 90% sequence identity' to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 95% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 96% sequence identity7to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 96% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 97% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having about 97% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 98% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5‘ UTR comprises a sequence having about 99% sequence identity' to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having about 99% sequence identity7to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 100% sequence identity' to SEQ ID NO: 21.

[0069] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 90% sequence identity' to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 90% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 95% sequence identity7to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 96% sequence identity to SEQ-40-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 96% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 97% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 97% sequence identity to SEQ ID NO: 6. In some embodiments, (I) the first 5’ UTR comprises a sequence having at least about 98% sequence identity7to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 98% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 99% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 96% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 97% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 97% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5?UTR comprises a sequence having at least 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least 99% sequence identity to SEQ ID NO: 6. In some embodiments. (1) the first 5?UTR comprises a sequence having about 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 80% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5‘ UTR comprises a sequence having about 90% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having about 90% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 95% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the-41-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOfirst 5’ UTR comprises a sequence having about 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 96% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 97% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having about 97% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5‘ UTR comprises a sequence having about 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 98% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5' UTR comprises a sequence having about 99% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having about 99% sequence identity’ to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having about 100% sequence identity’ to SEQ ID NO: 6.

[0070] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5‘ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 21. In some embodiments,(1) the first 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 2; and(2) the second 5‘ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 21. In some embodiments. (1) the first 5?UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 21.-42-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0071] In some embodiments, (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence of SEQ ID NO: 21.

[0072] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 70%. 72%. 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 21: and (2) the second 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 96% sequence identity’ to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5‘ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2. In some embodiments.(1) the first 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 21; and(2) the second 5’ UTR comprises a sequence having 98% sequence identity’ to SEQ ID NO: 2. In some embodiments, (1) the first 5‘ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 99% sequence identity' to SEQ ID NO: 2.

[0073] In some embodiments, (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence of SEQ ID NO: 2.

[0074] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity’ to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 6. In some-43-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, (1) the first 5’ UTR comprises a sequence having 95% sequence identify to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 95% sequence identify to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 96% sequence identify to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 96% sequence identify to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 97% sequence identify to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 97% sequence identify to SEQ ID NO: 6. In some embodiments,(1) the first 5’ UTR comprises a sequence having 98% sequence identify to SEQ ID NO: 2; and(2) the second 5’ UTR comprises a sequence having 98% sequence identify to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 99% sequence identify to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 99% sequence identify to SEQ ID NO: 6.

[0075] In some embodiments, (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence of SEQ ID NO: 6.

[0076] In some embodiments, the 5’ UTR overlaps with the core promoter. In some embodiments, the overlap is 7 nucleotides, wherein the 7 nucleotides are the 3’ 7 nucleotides of the core promoter. The core promoter can be a minP variant core promoter.

[0077] In some embodiments, the 5’ UTR is downstream of the promoter in the recombinant polynucleotide. In some embodiments, the 5’ UTR overlaps the 3’ end of the promoter. In some embodiments the 5’ UTR is upstream of the payload sequence.

[0078] The 5’ UTR may comprise a Kozak sequence. In some embodiments, the Kozak sequence is downstream of the promoter in the recombinant polynucleotide. In some embodiments the Kozak sequence is upstream of the payload sequence.Promoters

[0079] A polynucleotide (e.g., an RNA or a DNA polynucleotide) may comprise a 5 ' UTR of the present disclosure and a promoter sequence to regulate or enhance transcription of a payload, such as a transgene, in various target cell or tissue types, such as neural cell, retinal cell e.g.. retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell. A polynucleotide (e.g., an RNA or a DNA polynucleotide) may comprise a first 5’ UTR of the present disclosure and a first promoter sequence to regulate or enhance transcription of a pay load, such as a transgene, in various target cell or tissue types, such as neural cell, retinal cell e.g., retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone-44-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOcell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell, and a second 5’ UTR of the present disclosure and a second promoter sequence to regulate or enhance transcription of a payload, such as a transgene, in various target cell or tissue types, such as neural cell, retinal cell e.g., retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell. In some embodiments, the polynucleotide is a recombinant polynucleotide as described herein. In some embodiments, the promoter may comprise an enhancer and a core promoter sequence that functions as a site for preinitiation complex formation, or combinations thereof. In some embodiments, the promoter includes a core promoter and an enhancer. In some embodiments, the enhancer may comprise a transcription factor (TF) binding sequence that binds one or more transcription factors. In some embodiments, the core promoter may comprise a minimal synthetic promoter or a natural promoter. The elements within the promoter (e.g., the enhancer or the core promoter) may be selected or engineered to alter transcription rates of a downstream transgene. For example, the promoter may be selected to promote high levels of transcription in specific target cell or tissue types (e.g., RPE cells) and low levels or no transcription in non-target cell types. In some embodiments, the target tissue may be ocular / eye tissue (e.g.. ocular tissue comprising RPE), but can also include other tissue types depending on the therapeutic goal. In some embodiments, the target cells may be RPE cells. In some embodiments, the non-target tissue may be kidney tissue, muscle tissue, blood, skin, fat, bone, thymus tissue, gastrointestinal tissue (e.g., stomach, intestine), spleen tissue, placenta tissue, pancreatic tissue, lung tissue, liver tissue, or cardiac tissue. In some embodiments, the target tissue may be eye tissue (e.g., eye tissue comprising RPE cells) or any other tissue of therapeutic interest, and the non-target tissue may be kidney tissue, eye tissue, muscle tissue, blood, skin, fat, bone, thymus tissue, gastrointestinal tissue (e.g., stomach, intestine), spleen tissue, placenta tissue, pancreatic tissue, lung tissue, liver tissue, or cardiac tissue, or any combination thereof. In some embodiments, the non-target cell type may be renal cell, hepatocyte, podocyte, muscle cell, erythrocyte, platelet, bone marrow cell, endothelial cell, epidermal cell, lymphocyte, interstitial cell, adipocyte, or fibroblast. In some embodiments, the first promoter and the second promoter can be the same. In some embodiments, the first promoter and the second promoter can be different. In some embodiments, the polynucleotide comprises a first promoter and second promoter that are different and a first 5’ UTR and a second 5’ UTR that are different. In some embodiments, the polynucleotide comprises a first promoter and second promoter that are different and a first 5’ UTR and a second 5’ UTR that are the same. In some embodiments, the-45-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOpolynucleotide comprises a first promoter and a second promoter that are the same and a first 5’ UTR and a second 5’ UTR that are different. In some embodiments, the polynucleotide comprises a first promoter and a second promoter that are the same and a first 5’ UTR and a second 5’ UTR that are the same.Enhancers

[0080] The promoter of a polynucleotide may comprise an enhancer. The promoter of a polynucleotide may comprise an enhancer, wherein the polynucleotide comprises a 5’ UTR as described herein. The polynucleotide may be a recombinant polynucleotide as disclosed herein. An enhancer is any sequence that works to enhance the rate of transcription in one or more cell types. An enhancer paired with a core promoter as described herein may originally have been located upstream or downstream of and / or in the same or different orientation as a gene in any cell ty pe of interest. An enhancer paired with a core promoter as described herein may originally have been located in an exon or intron of and / or in the same or different orientation as a gene in any cell type of interest. The enhancer may recruit proteins to the polynucleotide that enhance, repress, or alter transcription of a dow nstream sequence (e.g., a transgene sequence encoded by the polynucleotide) in a cell-ty pe specific or more general manner. In some embodiments, an enhancer may bind transcription factors that promote transcription (e.g., by recruiting an RNA polymerase) of nearby coding sequences in specific cell types or across multiple cell types.

[0081] The enhancer may comprise a transcription factor binding sequence that binds one or more transcription factors specific to certain cell types or common across multiple cell types. The enhancer may comprise a portion of a transcription factor binding sequence that binds one or more transcription factors. The enhancer may comprise a motif of a transcription factor binding sequence that binds one or more transcription factors. The transcription factor binding sequence, the portion of the transcription factor binding sequence, or the motif of the transcription factor binding sequence may recruit one or more transcription factors to the polynucleotide that enhance, repress, or alter transcription of a downstream sequence (e.g.. a transgene sequence encoded by the polynucleotide) in various cellular contexts. The one or more transcription factors may be transcription factors expressed in specific cell types or more broadly across multiple cell types.

[0082] The polynucleotide may comprise an enhancer of a promoter and a 5' UTR as described herein. The polynucleotide may comprise a first enhancer of a first promoter as described herein and a second enhancer of a second promoter as described herein. The polynucleotide may comprise a first enhancer of a first promoter and a first 5’ UTR as disclosed herein and a second enhancer of a second promoter and a second 5’ UTR as disclosed herein.-46-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOCore Promoters

[0083] The promoter of a polynucleotide may comprise a core promoter. The promoter of a polynucleotide may comprise a core promoter, wherein the polynucleotide comprises a 5’ UTR as described herein. The polynucleotide may be a recombinant polynucleotide as disclosed herein. The promoter of a polynucleotide may comprise a core promoter that facilitates recruitment of transcription machinery and initiation of transcription in various cell types. In some embodiments, the core promoter may be positioned downstream (i.e., 3’) of an enhancer. In some embodiments, the core promoter may be positioned upstream (i.e., 5’) of a payload (e.g., a transgene). The core promoter may recruit polymerase or proteins that bind to polymerases to initiate transcription of a sequence downstream of the core promoter in a celltype specific or more general manner. For example, the core promoter may recruit an RNA polymerase (e.g., RNA polymerase II) or a TATA binding protein (TBP) that recruits an RNA polymerase. For example, the core promoter may bind to general transcription factors (GTFs) which recruit RNA polymerase II (Pol II) to initiate transcription. The ability of the core promoter to recruit transcription machinery (e.g., an RNA polymerase) or the affinity' of the core promoter for the transcription machinery may affect transcription levels across different cell types. In some embodiments, the core promoter may be altered to tune transcription levels by altering recruitment of and / or affinity for transcription machinery in specific cell types or more broadly.

[0084] A core promoter may be an endogenous promoter sequence. A core promoter sequence may be a synthetic promoter sequence. In some embodiments, the core promoter is a minimal synthetic core promoter. Core promoters may be selected or engineered for one or more desired transcriptional properties, such as transcription level, cell type specificity, or cell genotype specificity. For example, a core promoter may be selected to promote transcription in specific cell types such as RPE cells and little to no transcription other cell types. Engineering a core promoter may comprise screening variants of a core promoter for transcription level, cell type specificity, and / or cell genotype specificity in various cellular contexts.

[0085] In some embodiments, a core promoter may comprise a TATA box (e.g., TATAAA), an RNA polymerase binding sequence, a B recognition element (BRE, e.g., G / C,G / C,G / A,CGCC), a CCAAT box or CAT box (e.g., GGCCAATCT), or a Pribnow box (e.g., TATAAT)or a YB TATA promoter. Examples of core promoters are provided in TABLE 2.-47-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOTABLE 2 - Exemplary Core Promoters-48-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO-49-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO-50-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO-51-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO-52-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0086] In some embodiments, a core promoter may comprise a sequence of any of SEQ ID NO: 49-SEQ ID NO: 208. In some embodiments, the core promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 49- SEQ ID NO: 54. In some embodiments, the core promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55 to SEQ ID NO: 208. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 49. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 50. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 51. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 52. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 53. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 54. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 56. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 57. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 58. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 59. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 60. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 61. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 62. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 63. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 64. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 65. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 66. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 67. In some-53-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the core promoter may comprise a sequence of SEQ ID NO: 68. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 69. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 70. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 71. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 72. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 73. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 74. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 75. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 76. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 77. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 78. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 79. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 80. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 81. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 82. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 83. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 84. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 85. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 86. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 87. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 88. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 89. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 90. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 91. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 92. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 93. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 94. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 95. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 96. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 97. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 98. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 99. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 100. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 101. In some-54-KTS Ref.: 116779- 1521686-740WO1ShapeTX Ref. No.: STX-130WOembodiments, the core promoter may comprise a sequence of SEQ ID NO: 102. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 103. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 104. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 105. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 106. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 107. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 108. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 109. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 110. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 111. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 112. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 113. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 114. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 115. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 116. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 117. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 118. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 119. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 120. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 121. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 122. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 123. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 124. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 125. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 126. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 127. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 128. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 129. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 130. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 131. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 132. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 133. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 134. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 135. In some-55-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the core promoter may comprise a sequence of SEQ ID NO: 136. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 137. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 138. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 139. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 140. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 141. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 142. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 143. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 144. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 145. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 146. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 147. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 148. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 149. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 150. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 151. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 152. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 153. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 154. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 155. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 156. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 157. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 158. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 159. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 160. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 161. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 162. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 163. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 164. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 165. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 166. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 167. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 1 8. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 169. In some-56-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the core promoter may comprise a sequence of SEQ ID NO: 170. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 171. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 172. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 173. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 174. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 175. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 176. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 177. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 178. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 179. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 180. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 181. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 182. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 183. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 184. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 185. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 186. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 187. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 188. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 189. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 190. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 191. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 192. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 193. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 194. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 195. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 196. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 197. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 198. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 199. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 200. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 201. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 202. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 203. In some-57-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the core promoter may comprise a sequence of SEQ ID NO: 204. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 205. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 206. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 207. In some embodiments, the core promoter may comprise a sequence of SEQ ID NO: 208.

[0087] In some embodiments, the core promoter may be cell type generic. A cell type generic core promoter may have low basal activity alone (e.g., low levels of transcriptional activation in the absence of an enhancer) or when paired with an enhancer in a transcriptionally inactive state (e.g., an enhancer in the absence of co-activators, cognate ligands and / or transcription factors that bind to the enhancer) and high activity (e.g., high levels of transcriptional activation) when paired with an enhancer in a transcriptionally active state (e.g., an enhancer in the presence of co-activators, cognate ligands and / or transcription factors that bind to the enhancer). For example, a cell type generic core promoter may have low transcriptional activation in the absence of an enhancer, independent of cell or tissue type. The cell type generic core promoter may have high transcriptional activation when paired with a cell or tissue type-specific enhancer in a cell or tissue type of interest (e.g., in the presence of, or at high levels of, transcription factors that bind to the transcription factor binding sequence of an enhancer). The cell type generic core promoter may have low transcriptional activation when paired with a cell or tissue ty pe-specific enhancer not in a cell or tissue ty pe of interest (e g., in the absence of, or at low levels of, transcription factors that bind to the transcription factor binding sequence of an enhancer). In some embodiments, a core promoter may be engineered to have low basal transcriptional activation and high transcriptional activation when paired with a cell or tissue ty pe-specific enhancer in a cell or tissue type of interest. The sequence of the core promoter may be varied or engineered to tune the transcription level, tissue specificity', and / or cell type specificity. In some embodiments, a core promoter may comprise an endogenous core promoter or an engineered version of an endogenous core promoter.

[0088] The polynucleotide may comprise a core promoter of a promoter and a 5’ UTR as described herein. The polynucleotide may comprise a first core promoter of a first promoter as described herein and a second core promoter of a second promoter as described herein. The polynucleotide may comprise a first core promoter of a first promoter and a first 5‘ UTR as disclosed herein and a second core promoter of a second promoter and a second 5’ UTR as disclosed herein.

[0089] In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100%-58-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 70%. 72%. 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity’ to SEQ ID NO: 52 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity' to SEQ ID NO: 52 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 90% sequence identity to SEQ ID NO: 52 and further comprise the 5‘ UTR having 90% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 95% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 95% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 96% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 96% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 97% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 97% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 98% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 98% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 99% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter of SEQ ID NO: 52 and further comprise the 5’ UTR of SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 90% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 90% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 95% sequence identity to SEQ ID NO: 52 and further comprise the 5‘ UTR-59-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOhaving 95% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 96% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 96% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 97% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 97% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 98% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 98% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 99% sequence identity’ to SEQ ID NO: 52 and further comprise the 5?UTR having 99% sequence identity’ to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter of SEQ ID NO: 52 and further comprise the 5’ UTR of SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 90% sequence identity' to SEQ ID NO: 52 and further comprise the 5’ UTR having 90% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 95% sequence identity' to SEQ ID NO: 52 and further comprise the 5’ UTR having 95% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 96% sequence identity to SEQ ID NO: 52 and further comprise the 5‘ UTR having 96% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 97% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 97% sequence identity’ to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 98% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 98% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 99% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 99% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter of SEQ ID NO: 52 and further comprise the 5‘ UTR of SEQ ID NO: 6.

[0090] In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity' to SEQ ID NO: 53 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 53 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%,-60-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%, 80%, 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 70%, 72%, 75%, 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 90% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 90% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 95% sequence identity to SEQ ID NO: 53 and further comprise the 5‘ UTR having 95% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 96% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 96% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 97% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 97% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 98% sequence identity' to SEQ ID NO: 53 and further comprise the 5’ UTR having 98% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 99% sequence identity to SEQ ID NO: 52 and further comprise the 5’ UTR having 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter of SEQ ID NO: 53 and further comprise the 5’ UTR of SEQ ID NO: 2. In some embodiments, the polynucleotide may comprise a core promoter having 90% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 90% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 95% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 95% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 96% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 96% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 97% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 97% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 98% sequence identity-61-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOto SEQ ID NO: 53 and further comprise the 5' UTR having 98% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 99% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 99% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter of SEQ ID NO: 53 and further comprise the 5?UTR of SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise a core promoter having 90% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 90% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 95% sequence identity’ to SEQ ID NO: 53 and further comprise the 5?UTR having 95% sequence identity’ to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 96% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 96% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 97% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 97% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 98% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 98% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter having 99% sequence identity to SEQ ID NO: 53 and further comprise the 5’ UTR having 99% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise a core promoter of SEQ ID NO: 53 and further comprise the 5’ UTR of SEQ ID NO: 6.

[0091] In some embodiments, the polynucleotide may comprise (1) a first core promoter having 70%. 72%. 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity' to SEQ ID NO: 52 and (2) a second core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity' to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 80% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 80% sequence identity to SEQ ID NO: 53. and may further comprise (1) the first 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 90% sequence-62-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOidentity to SEQ ID NO: 52 and (2) a second core promoter having 90% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 95% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 95% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 96% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 96% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 97% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 97% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 98% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 98% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5‘ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 2; and (2) the second 5?UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 99% sequence identity7to SEQ ID NO: 52 and (2) a second core promoter having 99% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide may comprise (1) a first core promoter having of SEQ ID NO: 52 and (2) a second core promoter of SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence of SEQ ID NO: 21.

[0092] In some embodiments, the polynucleotide may comprise (1) a first core promoter having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 70%, 72%, 75%,-63-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 70%. 72%. 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 80% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 80% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 2; and (2) the second 5:UTR comprises a sequence having 80% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 90% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 90% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5' UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 95% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 95% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5' UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 96% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 96% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5‘ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 97% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 97% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 97% sequence identity7to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 98% sequence identity to SEQ ID NO: 52 and (2) a second core promoter having 98% sequence identity to SEQ ID NO: 53. and may further comprise (1) the first 5' UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having 99% sequence-64-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOidentity to SEQ ID NO: 52 and (2) a second core promoter having 99% sequence identity to SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 6. In some embodiments, the polynucleotide may comprise (1) a first core promoter having of SEQ ID NO: 52 and (2) a second core promoter of SEQ ID NO: 53, and may further comprise (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence of SEQ ID NO: 6.Payloads

[0093] A polynucleotide may comprise a 5’ UTR as described herein and a payload sequence. A polynucleotide may comprise a first 5’ UTR operably linked to a first payload sequence for expression of a first payload and second 5’ UTR as described operably linked to a second payload sequence for expression of a second payload. The polynucleotide may be a recombinant polynucleotide as disclosed herein. A payload of the present disclosure may comprise a sequence encoding a protein. In some embodiments, a payload as described herein is under transcriptional control of a promoter (e.g., a promoter comprising a enhancer and a core promoter). For example, a payload of the present disclosure may comprise a sequence encoding a protein under transcriptional control of an enhancer and a core promoter. The payload may comprise a transgene for delivery to a cell (e.g.. a cell of a human or non-human subject). In some embodiments, the transgene may comprise a coding sequence encoding a protein (e g., a protein without a mutation associated with a disease or condition). Upon delivery' of the payload to a cell, the protein encoded by the coding sequence may be expressed in the cell. In some embodiments, expression of a protein encoded by the coding sequence may treat, prevent, or alleviate symptoms of a disease or disorder. In some embodiments, the transgene may encode a wildtype copy of a protein that is mutated or dysregulated in the disease or condition.

[0094] Examples of genes that may be encoded by a payload sequence (e.g., the transgene) and delivered to a cell of a subject to treat, prevent, or alleviate symptoms of a disease or condition include MECP2. PRPH2, RHO, UBE3A, DYRK1A, MEF2C, NSD1. ATRX. RPS6KA3, TCF4, ZEB2, FOXG1, CDKL5, a partial piece of chromosome 2, SLC6A1, DMD, SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1 A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR, FLCN, FLCN, TP53. CRP. PTEN, IFT88, CLDN14. AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC 1, BAP1, APOE, CYBA, GSTT1, IFNG, IGF1, IL2, ABCB1, SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB, N0X4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT, CYBA, CYBB, EGFR, HM0X1, LRP2,-65-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOSERPINE1, PAX2, ABCB1, PPARA, PPARG, PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRAS1, CRB2, APRT, AXL, CCND1, CBR1, CPT1A, CYP1A1, CYP2B6, EDN1, ERBB2, HMGCR, MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2, PTGS2, HLTF, S0D1, SOD2, SREBF2, HNF1B, TERT. TNFSF10, NDRG1, MBTPS2, WNT4, BCOR, INF2, ALG9. BICC1, TMEM67. IRX2, FREM1, ANKS6. FREM2. CD46, COL4A3. COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9. Alternately or in addition, the payload sequence may encode a therapeutic polynucleotide that targets a gene, such as MECP2. PRPH2, RHO, UBE3A, DYRK1 A, MEF2C, NSD1, ATRX, RPS6KA3, TCF4, ZEB2, F0XG1, CDKL5. a partial piece of chromosome 2, SLC6A1, DMD. SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1 A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR, FLCN, FLCN, TP53, CRP, PTEN, IFT88, CLDN14, AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC1, BAP1, APOE, CYBA, GSTT1, IFNG. IGF1, IL2, ABCB1, SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB, N0X4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT, CYBA, CYBB, EGFR, HM0X1, LRP2, SERPINE1, PAX2, ABCB1, PPARA, PPARG, PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRAS1, CRB2, APRT, AXL, CCND1, CBR1, CPT1A. CYP1A1, CYP2B6, EDN1, ERBB2, HMGCR. MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2. PTGS2, HLTF. SOD1, SOD2, SREBF2, HNF1B, TERT, TNFSF10, NDRG1, MBTPS2, WNT4, BCOR, INF2, ALG9, BICC1, TMEM67, IRX2, FREM1, ANKS6, FREM2, CD46, COL4A3, COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9, to treat, prevent, or alleviate symptoms of a disease or condition associated with the gene.

[0095] In some embodiments, genes may be delivered as transgenes to a cell of a subject to treat a disease or condition in the subject. In some embodiments, the genes may be targeted by a protein (e.g., an antibody).

[0096] In some embodiments, the payload may comprise a therapeutic polynucleotide. In some embodiments, therapeutic polynucleotide encoded by the coding sequence may treat, prevent, or alleviate symptoms of a disease or disorder. In some embodiments, the payload may comprise a guide RNA sequence (e.g., for RNA or DNA editing), a tracrRNA, an siRNA, an shRNA, or a miRNA, an antisense oligonucleotide (e.g., for expression knockdown), a structural element (e.g., an RNA hairpin), or combinations thereof. In some embodiments, the payload may encode a guide RNA for adenosine deaminases acting on RNA (ADAR) editing. In some embodiments, the guide RNA may include a targeting sequence having sufficient complementarity to a target RNA to allow for hybridization of the targeting sequence to the target RNA. In some-66-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the targeting sequence has a minimum antisense complementarity of about 50, 60, 70, 80, 90, 100 or more nucleotides or more to the target RNA. In some embodiments, the guide RNA is 20 to 400 nucleotide residues long. In some embodiments, the guide RNA sequence is 50-200 nucleotide residues long. In some embodiments, the guide RNA sequence is 80-150 nucleotide residues long.

[0097] In some embodiments, the payload may be a tRNA targeting a gene associated with a disease or a condition. In some embodiments, the payload may be a tRNA designed to change the amino acid selected for protein synthesis. In some embodiments, the payload may be a tRNA designed to rescue a nonsense mutation which may result in premature stop codon.

[0098] In some embodiments, the payload may comprise polypeptides that form one or more functional antibodies or antibody-based compositions. Antibodies are primarily amino acid based molecules but may also comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.). Antibodies may be monomeric or multi-merit polypeptides which comprise at least one amino-acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence, e.g., mammalian protein. The encoded antibodies may be therapeutic, diagnostic, or for research purposes. Further, payloads described herein of the invention may include fragments of such antibodies or antibodies that have been developed to comprise one or more of such fragments (e.g., variable domains or complementarily determining regions (CDRs)) that include specific antigen-binding capability. Non-limiting examples of antibody fragments can include single-domain antibodies (VHHs or nanobodies), Fabs, F(ab’)2S, Fab2S (monospecific, bispecific, or trispecific), monovalent IgGs, scFvs, bispecific / bivalent scFvs (diabodies), triabodies, tetrabodies, scFv-Fcs, minibodies, single binding domains (dAbs), and minibodies. In some embodiments, the payload is a humanized antibody or humanized antibody fragment.

[0099] In some embodiments, a gene encoded by a payload (e.g., the transgene) may be expressed in a cell, such as neural cell, retinal cell e.g., retinal pigment epithelium (RPE) cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell at a level that is at least about 1-fold, at least about 1.1 -fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100--67-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOfold, at least about 150-fold, or at least about 200-fold higher expression level of the payload in a non-target cell type or tissue ty pe.

[0100] In some embodiments, the payload may be operably linked to a 5’ UTR e.g., any of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-24, that may increase payload expression in various cell types, with particular efficacy demonstrated in RPE cells, at a level that is at least about -0.75-fold, at least about -0.5-fold, at least about -0.25-fold, at least about -0.1 -fold, at least about O-fold, at least about 0.1-fold, at least about 0.25-fold, at least about 0.5-fold, at least about 0.75-fold, at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold, or at least about 200-fold compared to payload expression in other cell types. In some embodiments, the 5‘ UTR is SEQ ID NO: 2. In some embodiments, the 5’ UTR is SEQ ID NO: 21. While initial studies have focused on RPE cells, similar enhancement patterns may be observed in other specific cell types when compared to non-target cells. The degree of enhancement may vary depending on the specific 5 ' UTR sequence and the cellular context in which it is expressed.

[0101] In some embodiments, a gene encoded by a payload (e.g.. the transgene) may be expressed in various cell types, with particular focus on RPE cells, at a level that is at least about -0.75-fold, at least about -0.5-fold, at least about -0.25-fold, at least about -0.1-fold, at least about O-fold, at least about 0.1-fold, at least about 0.25-fold, at least about 0.5-fold, at least about 0.75-fold, at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold, or at least about 200-fold compared to the expression level of a wildtype version of the payload in the same cell type. While initial studies have concentrated on RPE cells, similar expression patterns may be observed in other specific cell types of interest. The level of expression may vary depending on the specific payload, the cellular context, and the regulatory elements used.Additional Elements

[0102] In some embodiments, the recombinant polynucleotide further comprises additional elements. In some embodiments, the recombinant polynucleotide comprises a 5’ UTR as described herein and further comprises additional elements. In some embodiments, the-68-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOrecombinant polynucleotide comprises a first 5’ UTR and a second 5’ UTR as described herein and further comprises additional elements. An additional element may enhance or increase expression of the payload, e.g., enhance or increase expression of a protein encoded by the payload. Examples of additional elements that may be included in a recombinant polynucleotide are a post-transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a CCCTC-binding factor sequence, a 3’ untranslated region, a 5’ stuffer sequence, and combinations thereof. For example, a recombinant polynucleotide comprises additional elements of a post-transcriptional regulatory element, a polyadenylation signal, and a transcriptional pause site. In another example, a post-transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a 3’ untranslated region, and a 5’ stuffer sequence.

[0103] In some embodiments, the recombinant polynucleotide comprises an element that increases expression of the payload, such as a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE) or a variant thereof (e.g., a shortened WPRE, WPRE3). In some embodiments, a WPRE comprises a sequence of GATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC CCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGA GTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAAC CCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTC CCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACA GGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCC TTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCT ACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGC CGCCTCCCCGCATCGG (SEQ ID NO: 214). In some embodiments, the WPRE has at least 80%. 90%. 95%. 96%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 214. In some embodiments, the recombinant polynucleotide is engineered to comprise a polyadenylation (poly A) signal for increased expression of the pay load. For example, the recombinant polynucleotide is engineered to comprise a rabbit beta globulin polyA sequence. As another example, the recombinant polynucleotide is engineered to comprise a bovine growth hormone (bGH) polyA sequence. In some embodiments, a polyA sequence comprises a sequence of cgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca ctgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagg-69-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOgggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctcgac tagagcttgcggaaccctta (SEQ ID NO: 215). In some embodiments, the polyA sequence has at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 215. In some embodiments the recombinant polynucleotide is engineered to comprise an element that reduces transcriptional silencing, such as a transcriptional pause site. In some embodiments, a transcriptional pause site may comprise a polyA signal (e.g., a second polyA signal). The transcriptional pause site may reduce transcriptional silencing caused by transcriptional readthrough from an upstream promoter.

[0104] In some embodiments, an additional element is a CCCTC-binding factor (CTCF) sequence. A CTCF sequence may modify accessibility of the recombinant polynucleotide to transcription machinery to enhance expression of the pay load. The CTCF sequence may be positioned in the recombinant polynucleotide for payload expression such that the CTCF modifies accessibility of the recombinant polynucleotide to transcription machinery to enhance expression of the pay load.

[0105] In some embodiments, an additional element is a 3’ untranslated region (3’ UTR). The 3’ UTR may be positioned in the recombinant polynucleotide such that the 3’ UTR regulates payload expression. The polyA signal sequence may regulate expression of the payload.

[0106] In some embodiments, the recombinant polynucleotide further comprises a post- transcriptional regulatory element. In some embodiments, a post-transcriptional regulatory element is included in a 3 ’untranslated region of a recombinant polynucleotide. A post- transcriptional regulatory element may enhance expression of a payload (e.g., a coding sequence encoding a protein or a polynucleotide) of a recombinant polynucleotide.

[0107] In some embodiments, the post-transcriptional regulatory element is part of a 3’ untranslated region (3’ UTR). In some embodiments, a 3’ UTR comprises a post-transcriptional regulator}7element.

[0108] In some embodiments, the WPRE element is a WPRE variant. In some embodiments, the WPRE variant is a shortened WPRE or truncated WPRE. A shortened WPRE or truncated WPRE (e.g., a WPRE3) may exhibit similar transcriptional activity as a full length WPRE. A shorted WPRE or truncated WPRE may be preferred over a full length WPRE due to its shorter nucleotide sequence. In some embodiments, the shortened WPRE is a WPRE3. In some embodiments, the recombinant polynucleotide further comprises a WPRE3. In some embodiments, the WPRE3 is downstream of the promoter in the recombinant polynucleotide. In some embodiments the WPRE3 is downstream of the payload sequence. In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 WPRE3s.-70-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0109] In some embodiments, the recombinant polynucleotide further comprises a polyadenylation (poly A) signal. The polyA signal may signal formation of a poly A tail to an RNA transcribed from the recombinant polynucleotide. In some embodiments, the polyA signal is downstream of the promoter in the recombinant polynucleotide. In some embodiments the polyA signal is downstream of the payload sequence. In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 polyA signals.

[0110] In preferred embodiments, the polyA signal is a rabbit beta globin polyA signal. In some embodiments, the rabbit beta globin polyA signal is downstream of the promoter in the recombinant polynucleotide. In some embodiments the rabbit beta globin polyA signal is downstream of the pay load sequence. In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 rabbit beta globin polyA signals.[OHl] In some embodiments, the polyA signal is part of a 3’ untranslated region (3’ UTR). In some embodiments, a 3' UTR comprises a polyA signal.

[0112] In some embodiments, the recombinant polynucleotide further comprises a transcriptional pause site. The transcriptional pause site may facilitate transcriptional termination and prevent transcriptional interference. The transcriptional pause site may reduce transcriptional silencing for example, transcriptional silencing caused by transcriptional readthrough from an upstream promoter. In some embodiments, the transcriptional pause site includes a second polyA signal. In some embodiments, the transcriptional pause site is dow nstream of the promoter in the recombinant polynucleotide. In some embodiments the transcriptional pause site is dow nstream of the payload sequence.

[0113] In some embodiments, the recombinant polynucleotide further comprises a stuffer sequence. In some embodiments, the stuffer sequence is upstream of the promoter in the recombinant polynucleotide. In some embodiments the stuffer sequence is upstream of the payload sequence. In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or stuffer sequences.

[0114] In some embodiments, the recombinant polynucleotide further comprises a 5’ inverted terminal repeat (5’ ITR). The 5’ ITR may be selected based on a vector, such as a viral vector, used to deliver the recombinant polynucleotide. In some embodiments, the 5‘ ITR may be an AAV 5’ ITR (e g., an AAV5 5’ ITR, an AAV1 5’ ITR, an AAV2 5’ ITR, an AAV9 5’ ITR, or a PhP.eB 5’ ITR). In some embodiments, the 5’ ITR is upstream of the promoter in the recombinant polynucleotide. In some embodiments the 5’ ITR is upstream of the pay load sequence. In some embodiments, the recombinant polynucleotide comprises at least 1, 2, 3, 4, or 5’ ITRs.-71-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0115] In some embodiments, the recombinant polynucleotide further comprises a 3’ inverted terminal repeat (3’ ITR). The 3’ ITR may be selected based on a vector used to deliver the recombinant polynucleotide. In some embodiments, the 3’ ITR may be an AAV 3’ ITR (e.g., an AAV5 3’ ITR, an AAV1 3’ ITR, an AAV2 3’ ITR, an AAV9 3’ ITR, or a PhP.eB 3’ ITR). In some embodiments, the 3’ ITR is downstream of the promoter in the recombinant polynucleotide. In some embodiments the 3’ ITR is downstream of the pay load sequence. In some embodiments, the recombinant polynucleotide comprises at least 1, 2, 3, 4, or 3’ ITRs.

[0116] In some embodiments, a recombinant polynucleotide includes one or more cloning sites. A cloning site may include a restriction site that may be specifically cut by a restriction enzyme. In some embodiments, a cloning site is a multiple cloning site comprising multiple restriction sites (e.g., from 2 to 20 restriction sites). A cloning site may be included between sequence elements within a recombinant polynucleotide (e.g., between a coding sequence and an additional element, between a coding sequence and a promoter, between a promoter and an additional element, between two additional elements, or combinations thereol). A cloning site can comprise a restriction enz me cut site (also referred to as a “cut site” or a “restriction site”). Examples of cloning sites that may be included in a recombinant polynucleotide of the present disclosure are a Hindlll cut site, a Xbal cut site, aNotl cut site, a Sall cut site, a PstI cut site, and combinations thereof. In some embodiments, a recombinant polynucleotide includes a cloning site between a coding sequence and a post-transcriptional regulatory element. For example, a recombinant polynucleotide includes a Hindlll cut site between a payload sequence and a WPRE3 post-transcriptional regulatory element. In some embodiments, a recombinant polynucleotide includes a cloning site between a post-transcriptional regulatory’ element and a polyadenylation site.Recombinant Vectors

[0117] In some embodiments, the polynucleotide of the present disclosure is introduced into a subject via a recombinant vector. In some embodiments, the polynucleotide is a recombinant polynucleotide of the present disclosure. In some embodiments, the polynucleotide comprises a 5’ UTR of the present disclosure. In some embodiments, the polynucleotide comprises a first 5’ UTR and a second 5’ UTR of the present disclosure. In some embodiments the vector is a plasmid, a viral vector, an expression cassette, or a transformed cell. The compact size of the enhancers described herein may facilitate incorporation into a recombinant vector, allowing for inclusion of larger payloads.

[0118] In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector. Adeno-associated virus (AAV) vectors include vectors derived from-72-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOany AAV serotype, including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAVrh8, AAV.rhlO, AAVrh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAVOligoOOl, AAVSCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAVAnc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHPB, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAV.HSC1, AAVHSC2, AAV.HSC3, AAVHSC4, AAV.HSC5, AAVHSC6, AAV.HSC7, AAV.HSC8, AAVHSC9, AAV.HSC10, AAVHSCH, AAVHSC12, AAVHSC13, AAVHSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, and AAVhu68.

[0119] In some embodiments, a polynucleotide is introduced into a subject by non-viral vector systems. In some embodiments, cationic lipids, polymers, hydrodynamic injection and / or ultrasound may be used in delivering a polynucleotide to a subject in the absence of virus.

[0120] In some examples, the vector may be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof. In some examples, the vector may be a viral vector. In some embodiments, the viral vector may be a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a lentivirus vector (e.g., human or porcine), a Herpes virus vector, an Epstein-Barr virus vector, an SV40 virus vectors, a pox virus vector, or a combination thereof. In some embodiments, the viral vector may be a recombinant vector, a hybrid vector, a chimeric vector, a self-complementary vector, a singlestranded vector, or any combination thereof.

[0121] In some embodiments, the viral vector may be an adeno-associated virus (AAV). In some embodiments, the AAV may be any AAV known in the art. In some embodiments, the viral vector may be of a specific serotype. In some embodiments, the viral vector may be an AAV 1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV 10 seroty pe, AAV 11 serotype, AAV 12 serotype, AAV13 serotype. AAV14 serotype, AAV15 serotype, AAV16 serotype, AAV-DJ serotype. AAV-DJ / 8 serotype, AAV-DJ / 9 serotype, AAV 1 / 2 serotype. AAV.rh8 serotype, AAV.rhlO serotype, AAV.rh20 serotype, AAV.rh39 serotype, AAV.Rh43 serotype, AAV.Rh74 serotype, AAV.v66 serotype, AAVOligoOOl serotype, AAV.SCH9 serotype, AAV.r3.45 serotype, AAV.RHM4-1 serotype, AAV.hu37 serotype, AAV. Anc80 serotype, AAV. Anc80L65 serotype, AAV.7m8 serotype, AAV.PhP.eB serotype. AAV.PhP.Vl serotype. AAV.PHP.B serotype, AAV.PhB.Cl serotype, AAV.PhB.C2 serotype, AAV.PhB.C3 serotype, AAV.PhB.C6 serotype, AAV.cy5 serotype, AAV2.5 serotype, AAV2tYF serotype, AAV3B serotype, AAV.LK03 serotype, AAV.HSC1 serotype, AAV.HSC2 serotype, AAV.HSC3 serotype, AAV.HSC4 serotype,-73-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOAAV.HSC5 serotype, AAV.HSC6 serotype, AAV.HSC7 serotype, AAV.HSC8 serot pe. AAV.HSC9 serotype, AAV.HSC10 serotype, AAV.HSC11 serotype, AAV.HSC12 serotype, AAV.HSC13 serotype, AAV.HSC14 serotype, AAV.HSC15 serotype, AAV.HSC16 serotype, AAV.HSC17 serotype, or AAVhu68 serotype, a derivative of any of these serotypes, or any combination thereof.

[0122] In some embodiments, the AAV vector may be a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof.

[0123] In some embodiments, the AAV vector may be a recombinant AAV (rAAV) vector or engineered AAV vector. Methods of producing recombinant AAV vectors may be known in the art and generally involve, in some cases, introducing into a producer cell line: (1) DNA necessary for AAV replication and synthesis of an AAV capsid, (b) one or more helper constructs comprising the viral functions missing from the AAV vector, (c) a helper virus, and (d) the plasmid construct containing the genome of the AAV vector, e.g., ITRs, promoter and transgene sequences, etc. In some examples, the viral vectors described herein may be engineered through synthetic or other suitable means by references to published sequences, such as those that may be available in the literature. For example, the genomic and protein sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits may be known in the art and may be found in the literature or in public databases such as GenBank or Protein Data Bank (PDB).

[0124] An AAV vector may be assembled from one or more viral capsid proteins (VP). Viral capsid protein is referred to as AAV5 VP1 when referencing AAV5 VP1 positional notation. In all cases, viral capsid sequences and mutations disclosed herein should be understood as pertaining to all isoforms of the capsid protein (VP1, VP2, and VP3), as a mixture of these isoforms assemble to form virions. The positional amino acid residue designations “581 to 589” are relative to the translational start of the AAV 5 VP1 polypeptide and should be adjusted accordingly to the relative start sites of AAV5 VP2 and AAV5 VP3. It should be understood that the present disclosure, when describing any particular VP1 sequence with mutations at particular amino acid residue positions, necessarily also encompasses corresponding mutations in VP2 and VP3. For example, any consensus sequence or specific sequence of an AAV5 VP1 capsid protein having one or more mutations in the 581-589 region, corresponding to amino acid residues 581 to 589 of AAV 5 VP1, also encompasses AAV 5 VP2 and AAV 5 VP3 capsid proteins having said one or more mutations in an amino acid residue region in VP2 and VP3 corresponding to the amino acid residues of the VP1 581 to 589 region. For example, the amino acid residues of the-74-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO581 to 589 region of AAV5 VP1 (SEQ ID NO: 209);MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGL DRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGK AVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGS QQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVT KSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQ RLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGN GTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERS SFFCLEYFP SKMLRTGNNFEFTYN FEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFP GPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYAL ENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATG TYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTP VPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDF APDSTGEYRTTRPIGTRYLTRPL) correspond to the amino acid residues of the 445 to 453 region of AAV5 VP2 (SEQ ID NO: 210);TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAG GGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKS GSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQ VKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYA TLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPL VDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASV SAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEG NMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYL QGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYST GQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRP L) and to the amino acid residues of 389 to 397 region of AAV5 VP3 (SEQ ID NO: 211);MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQY REIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVK IFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQ YGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKL ANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVN RASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTAT YLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERD VYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQ-75-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRY LTRPL). As used herein, “wild type AAV5” or “wild type AAV5 capsid polypeptide” refers to an AAV5 VP1 capsid polypeptide of SEQ ID NO: 209, an AAV5 VP2 capsid polypeptide of SEQ ID NO: 210, an AAV5 VP3 capsid polypeptide of SEQ ID NO: 211, or a combination thereof. Also as used herein, a “wild type 581-589 region” refers to a 581 to 589 region of AAV5 VP1 having a sequence of ATGTYNLQE (SEQ ID NO: 213).

[0125] As used herein, “581-589 region” refers to a region or fragment of AAV5 VP1 corresponding to amino acid residues 581 to 589 relative to the translational start of the AAV5 VP1 polypeptide. A 581-589 region comprising at least one mutation relative to a wild type 581- 589 region sequence (e.g., a 581-589 region of a wild type AAV5 VP1 capsid polypeptide) may also be referred to as a “variant region” or a “variant 581-589 region.” The 581-589 region corresponds to amino acid residues 445 to 453 of AAV5 VP2 and to amino acid residues 389 to 397 of AAV5 VP3. The 581-589 region may confer tissue tropism to an AAV, and defined variants may be engineered to confer tissue tropism to an rAAV formed from viral capsid polypeptides (VP1, VP2, and VP3) comprising the 581-589 region. The AAV5 VP1 with a generalized 581-589 region is provided in SEQ ID NO: 212 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGK AVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGS QQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVT KSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQ RLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGN GTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYN FEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFP GPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYAL ENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPX’X2X3X4X5X6X7X8X9IVPGSVWMERDVYLQGPIWAK1PETGAHFHPSPAMGGFGLKHPPPMM LIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDP QFVDFAPDSTGEYRTTRPIGTRYLTRPL), in which the 581-589 region has a sequence of X1X2X3X4X5X6X7X8X9; wherein X1, X2, X3, X4, X5, X6, X7, X8, and X9are each independently selected from A, R, N, D, C. E, Q. G, H. I, L, K, M. F, P, S, T. W, Y, and V.

[0126] In some embodiments, an engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide has an amino acid sequence at least 70% identical to an AAV5 VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID-76-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WONO: 211), wherein the engineered AAV 5 VP capsid polypeptide has at least one substitution as compared to SEQ ID NO: 209 in the 581-589 region, corresponding to residue 581 to residue 589 of SEQ ID NO: 209, inclusive, wherein the capsid polypeptide is capable of assembling into a recombinant AAV5 virion (rAAV5). In some embodiments, the AAV5 VP capsid polypeptide comprises a variant 581-589 region (e.g., comprising one or more amino acid substitutions relative to SEQ ID NO: 209 in the region from residue 581 to residue 589, inclusive). The AAV5 VP capsid polypeptide may comprise a sequence of SEQ ID NO: 212, wherein the 581-589 region has a one or more mutations relative to the wild type AAV5 VP1 capsid polypeptide, wherein the one or more mutations confers tissue tropism (e.g., RPE tropism).

[0127] In particular embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide has an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to an AAV5 VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID NO: 211.

[0128] In some embodiments, the AAV VP capsid polypeptides have an amino acid sequence of SEQ ID NO: 212, wherein X1, X2, X3, X4, X5, X6, X7, X8, and X9are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V

[0129] In some examples, methods of producing delivery vectors herein comprising packaging a polynucleotide of the present disclosure in an AAV vector (e.g.. a recombinant AAV 5 comprising an engineered AAV5 VP capsid polypeptide). In some examples, methods of producing the delivery vectors described herein comprise, (a) introducing into a cell: (i) a polynucleotide disclosed herein; and (ii) a viral genome comprising a Replication (Rep) gene and Capsid (Cap) gene that encodes a wild-type AAV capsid protein or modified version thereof; (b) expressing in the cell the wild-type AAV capsid protein or modified version thereof; (c) assembling an AAV particle; and (d) packaging the polynucleotide disclosed herein in the AAV particle, thereby generating an AAV delivery' vector. In some examples, any polynucleotide disclosed herein may be packaged in the AAV vector. In some examples, the recombinant vectors comprise one or more inverted terminal repeats and the inverted terminal repeats comprise a 5’ inverted terminal repeat, a 3’ inverted terminal repeat, and a mutated inverted terminal repeat. In some examples, the mutated terminal repeat lacks a terminal resolution site, thereby enabling formation of a self-complementary' AAV.

[0130] In some examples, a hybrid AAV vector may be produced by transpeptidation, e.g.. packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) may be used in a capsid from a-77-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsecond AAV serotype (e.g., AAV 5 or AAV9), wherein the first and second AAV serotypes may not be the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2 / 9. In some examples, the hybrid AAV delivery vector comprises an AAV2 / 1, AAV2 / 2, AAV 2 / 4, AAV2 / 5, AAV2 / 8. or AAV2 / 9 vector.

[0131] In some examples, the AAV vector may be a chimeric AAV vector. In some examples, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.

[0132] In some examples, the AAV vector comprises a self-complementary AAV genome. Self- complementary AAV genomes may be generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA

[0133] In some examples, the delivery vector may be a retroviral vector. In some examples, the retroviral vector may be a Moloney Murine Leukemia Virus vector, a spleen necrosis virus vector, or a vector derived from the Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, or mammary tumor virus, or a combination thereof. In some examples, the retroviral vector may be transfected such that the majority of sequences coding for the structural genes of the virus (e.g., gag, pol, and env) may be deleted and replaced by the gene(s) of interest.

[0134] In some examples, the delivery vehicle may be a non-viral vector. In some examples, the delivery vehicle may be a plasmid. In some embodiments, the plasmid may be pGL4. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some examples, the plasmid comprises circular double-stranded DNA. In some examples, the plasmid may be linear. In some examples, the plasmid comprises one or more genes of interest and one or more regulatory elements. In some examples, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the plasmid may be a minicircle plasmid. In some examples, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid may be formulated for delivery through injection by a needle cartying syringe. In some examples, the plasmid may be formulated for delivery via electroporation. In some examples, the plasmids may be engineered through synthetic or other suitable means known in the art. For example, in some cases, the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.-78-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOMethods of Regulating Payload Translation

[0135] In some embodiments, the present disclosure provides a method of inserting a polynucleotide comprising the 5’ UTR as described herein and the pay load into a recombinant polynucleotide cassette. In some embodiments, the present disclosure provides a method of inserting a polynucleotide comprising the first 5’ UTR as described herein and the first payload and the second 5’ UTR as described herein and the second payload into a recombinant polynucleotide cassette. In some embodiments, the present disclosure provides a method of inserting a polynucleotide comprising the promoter as described herein and the payload into a recombinant polynucleotide cassette. The recombinant polynucleotide cassette may further modulate expression of the payload (e.g., by modulating translation). In some embodiments, the recombinant polynucleotide cassette modulates stability' of the pay load RNA. In some embodiments, the recombinant polynucleotide cassette comprises a 5’ UTR. In some embodiments, the recombinant polynucleotide cassette comprises a first 5' UTR and a second 5‘ UTR. In some embodiments, the recombinant polynucleotide cassette comprises a 3’ UTR. In some embodiments, the payload is codon optimized in the recombinant polynucleotide cassette. In some embodiments, an intron is inserted into the 5‘ UTR or the sequence of the payload. In some embodiments, the intron is a natural intron or a synthetic intron.

[0136] In some embodiments, the recombinant polynucleotide cassette comprises the promoter as described herein, the payload, and one or more of: a 5’ UTR region; a 3’ UTR region; a codon optimized sequence of the pay load; and an intron in the sequence of the pay load. In some embodiments, translation of the payload increases from the recombinant polynucleotide cassette comprising one or more of the 5’ UTR region, the 3?UTR region, a codon optimized sequence of the payload, and the intron in the sequence of the pay load compared to from a recombinant polynucleotide cassette lacking the 5’ UTR region, the 3’ UTR region, a codon optimized sequence of the pay load, and the intron in the sequence of the pay load. In some embodiments, translation of the payload decreases from the recombinant polynucleotide cassette comprising one or more of the 5’ UTR region, the 3‘ UTR region, a codon optimized sequence of the pay load, and the intron in the sequence of the pay load compared to from a recombinant polynucleotide cassette lacking the one or more of the 5’ UTR region, the 3’ UTR region, a codon optimized sequence of the payload, and the intron in the sequence of the payload.

[0137] In some embodiments, the 5’ UTR may comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or-79-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOabout 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the 5’ UTR may comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%. at least about 85%, at least about 87%, at least about 90%, at least about 92%. at least about 93%. at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%. at least about 80%, at least about 82%, at least about 85%, at least about 87%. at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%. at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 96% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 97% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 96% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 97% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 99% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having at least about 90% sequence identity to SEQ ID NO: 6. In some embodiments, the-80-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO5’ UTR may comprise a sequence having at least about 95% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 96% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 97% sequence identity to SEQ ID NO: 6. In some embodiments, the 5‘ UTR may comprise a sequence having at least about 98% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having at least about 99% sequence identity' to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 6. In some embodiments, the 5' UTR may comprise a sequence having 70%. 72%. 75%. 78%. 80%. 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity’ to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the 5’ UTR may comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the 5' UTR may comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the 5' UTR may comprise a sequence having 90% sequence identity’ to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 95% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 96% sequence identity to SEQ ID NO: 2. In some embodiments, the 5‘ UTR may comprise a sequence having 97% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 98% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 99% sequence identity to SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 2. In some embodiments, the 5’ UTR may comprise a sequence having 90% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 95% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 96% sequence identity’ to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 97% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 98% sequence identity to SEQ ID NO: 21. In some embodiments, the 5?UTR may comprise a sequence having 99% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 21. In some embodiments, the 5’ UTR may comprise a sequence having 90% sequence identity’ to SEQ ID NO: 6. In some-81-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOembodiments, the 5’ UTR may comprise a sequence having 95% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 96% sequence identity to SEQ ID NO: 6. In some embodiments, the 5' UTR may comprise a sequence having 97% sequence identity to SEQ ID NO: 6. In some embodiments, the 5‘ UTR may comprise a sequence having 98% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence having 99% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ UTR may comprise a sequence of SEQ ID NO: 6.

[0138] In some embodiments, the recombinant polynucleotide comprises one 5’ UTR. In some embodiments, the recombinant polynucleotide comprises two 5?UTRs (e.g., a first 5?UTR and a second 5’ UTR) and the two 5’ UTRs have the same sequence. In some embodiments, the two 5’ UTRs (e.g., the first 5’ UTR and the second 5’ UTR) each comprise a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%. at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24. In some embodiments, the two 5‘ UTRs (e.g., the first 5' UTR and the second 5’ UTR) may each comprise a sequence having 70%, 72%, 75%, 78%, 80%, 82%. 85%. 87%. 90%. 92%. 93%. 95%. 97%. 98%. 99%. or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24.

[0139] In some embodiments, the recombinant polynucleotide comprises two different 5’ UTRs. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%. at least about 78%. at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; (2) the second 5‘ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; and the first and the second 5?UTRs have different sequences (e.g., different SEQ ID NOs).

[0140] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence-82-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOidentity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9 and SEQ ID NO: 12-SEQ ID NO: 24; and (3) and the first and the second 5‘ UTRs have different sequences (e.g., different SEQ ID NOs). In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%. at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%. at least about 99%. or about 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9; and (2) the second 5’ UTR comprises a sequence having at least about 70%, at least about 72%, at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%. at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24.

[0141] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%. 99%. or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 9; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24.

[0142] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%. at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%. at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NO: 12- SEQ ID NO: 24.

[0143] In some embodiments, (1) the first 5?UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity7to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%,-83-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 12-SEQ ID NO: 24.

[0144] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%. at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%. at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21.

[0145] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%. at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2.

[0146] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 70%. at least about 72%. at least about 75%, at least about 78%, at least about 80%, at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 70%, at least about 72%. at least about 75%, at least about 78%, at least about 80%. at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 6.

[0147] In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 90% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 95% sequence identity-84-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOto SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 96% sequence identity to SEQ ID NO: 21. In some embodiments,(1) the first 5’ UTR comprises a sequence having at least about 97% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 97% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 98% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having at least about 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having at least about 99% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 90% sequence identity7to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 21. In some embodiments. (1) the first 5?UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2; and (2) the second5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 21. In some embodiments, (1) the first 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 21. In some embodiments. (1) the first 5?UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 21.

[0148] In some embodiments, (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 2; and(2) the second 5’ UTR comprises a sequence of SEQ ID NO: 21.

[0149] In some embodiments, (1) the first 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 70%, 72%,-85-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5?UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 21; and (2) the second 5' UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2. In some embodiments, (1) the first 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 21; and (2) the second 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 2. In some embodiments,(1) the first 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 21; and(2) the second 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 2.

[0150] In some embodiments, (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 21; and (2) the second 5‘ UTR comprises a sequence of SEQ ID NO: 2.

[0151] In some embodiments, (1) the first 5?UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity7to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 70%, 72%, 75%, 78%, 80%, 82%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5?UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 90% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 95% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 96% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence having 97% sequence identity to SEQ ID NO: 6. In some embodiments, (1) the first 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 2; and (2) the second 5’ UTR comprises a sequence having 98% sequence identity to SEQ ID NO: 6. In some embodiments,-86-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO(1) the first 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 2; and(2) the second 5’ UTR comprises a sequence having 99% sequence identity to SEQ ID NO: 6.

[0152] In some embodiments, (1) the first 5’ UTR comprises a sequence of SEQ ID NO: 2; and (2) the second 5‘ UTR comprises a sequence of SEQ ID NO: 6.

[0153] In some embodiments, the 5’ UTR region comprises one or more of: a structural element; a sequence motif: a nucleotide base content comprising a G / C content of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100%; and a 5’ UTR intron. In some embodiments, the structural element is a site that is in a conformation with the 5’cap so that the 5’cap is inaccessible or has low accessibility to the translation machinery (e.g., also referred to as “a cap-burying site”), resulting in decreased or no translation compared to a 5’ UTR lacking this structural element. In some embodiments, the structural element is an Internal Ribosome Entry Site (IRES). In some embodiments, the structural element is an RNA pseudoknot. In some embodiments, the structural element is an Iron Responsive Element (IRE). In some embodiments, the structural element is a non-coding translation modulatory structure. In some embodiments, the structural element is a hairpin. In some embodiments, the structural element is a sequence (e.g., a cap-burying site sequence, an IRES, an RNA pseudoknot, an IRE, or a non-coding translation modulatory structure), that changes conformation when a sequence element contacts a target RNA with which it has at least partial complementarity, such that the rate of translation of the payload downstream of the structural element is increased or decreased compared to translation of the payload prior to the sequence element contacting the target RNA. In some embodiments, the 5’ UTR region further comprises the sequence element, wherein a nucleic acid sequence of the sequence element is at least partially complementary to a sequence of a target RNA and wherein conformation of the structural element changes when the sequence element contacts the target RNA with which it has at least partial complementarity7. In some embodiments, the 5‘ UTR intron is a natural intron, synthetic intron, or a fragment thereof.

[0154] In some embodiments, the 3’ UTR comprises one or more of: a site that recruits polyA tail machinery; an miRNA binding site; or a sequence motif. In some embodiments, the poly(A) tail recruitment machinery7comprises an enzyme. In some embodiments, the length of the poly(A) tail modulates protein expression from the polynucleotide. In some embodiments, the 3’ UTR region comprises one. two, three, four, five, six, seven, eight, nine, ten, or more miRNA binding sites. In some embodiments, the miRNA binding sites are for the same miRNA. In some embodiments, the miRNA binding sites are for different miRNA. In some embodiments, the-87-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOsequence motif is an AC-rich motif. In some embodiments, the sequence motif is an AU-rich element (ARE).

[0155] In some embodiments, the codon optimized sequence of the therapeutic polynucleotide is a least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100% codon optimized. In some embodiments, the intron in the sequence of the therapeutic polynucleotide is a natural intron, synthetic intron, or a fragment thereof. In some embodiments, the recombinant polynucleotide cassette is encoded by a DNA vector.

[0156] In some embodiments, the combination of the promoter and pay load as described herein in the recombinant polynucleotide cassette results in the payload being expressed at a therapeutic level for reducing or alleviating at least one symptom of the disease or disorder. The therapeutic level can be -0.25-fold, -0.5-fold, O-fold, 0.25-fold, 0.5-fold, 0.75-fold, 1-fold, 1.5- fold, 2-fold, or 4-fold greater than the biological level of the payload.

[0157] In some embodiments, the payload is expressed at a level that is from about 1.5-fold to about 9-fold higher than a control level of pay load expression from inserting an otherwise comparable recombinant polynucleotide lacking the 5’ UTR into the cell. In some embodiments, the payload is expressed a level that is about 2-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 3-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 4-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 5-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 6-fold higher than the control level of pay load expression. In some embodiments, the payload is expressed at a level that is about 7-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 8-fold higher than the control level of payload expression. In some embodiments, the payload is expressed at a level that is about 9-fold higher than the control level of payload expression.Methods of Treatment and Pharmaceutical Compositions

[0158] Methods for treatment of diseases or disorders characterized by aberrant gene expression are also encompassed by the present disclosure. Said methods include administering a therapeutically effective amount of a transgene as part of a recombinant polynucleotide cassette. Said methods include administering a therapeutically effective amount of a trans gene as part of a recombinant polynucleotide cassette that comprises a 5’ UTR of the present disclosure. Said methods include administering a therapeutically effective amount of a first transgene and a second transgene as part of a recombinant polynucleotide cassette that comprises a first 5’ UTR-88-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOand second 5’ UTR of the present disclosure. The recombinant polynucleotide cassette of the disclosure can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the recombinant polynucleotide cassettes, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0159] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder, or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0160] For intravenous, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required.

[0161] In some embodiments, the polynucleotide of the present disclosure or recombinant polynucleotide cassette of the present disclosure may be administered to cells via a lipid nanoparticle. In some embodiments, the lipid nanoparticle may be administered at the appropriate concentration according to standard methods appropriate for the target cells.

[0162] In some embodiments, the polynucleotide of the present disclosure or recombinant polynucleotide cassette of the present disclosure may be administered to cells via a viral vector. In some embodiments, the viral vector may be administered at the appropriate multiplicity of infection according to standard transduction methods appropriate for the target cells. Titers of the virus vector or capsid to administer can vary depending on the target cell type and number and can be determined by those of skill in the art. In some embodiments, at least about 102infections units are administered. In some embodiments, at least about 103. 104, 10’. 106. 107, 108, 109, 1010, 1011, 1012, or 1013infectious units are administered.

[0163] In some embodiments, the polynucleotide or the disclosure or the recombinant polynucleotide cassette of the disclosure may be introduced to cells in vitro via a viral vector for-89-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOadministration of modified cells to a subject. In some embodiments, a viral vector encoding the polynucleotide of the disclosure, or the recombinant polynucleotide cassette of the disclosure is introduced to cells that have been removed from a subject. In some embodiments, the modified cells are placed back in the subject following introduction of the viral vector.

[0164] In some embodiments, a dose of modified cells is administered to a subject according to the age and species of the subject, disease or disorder to be treated, as well as the cell type and mode of administration. In some embodiments, at least about 102- 108cells are administered per dose. In some embodiments, cells transduced with viral vector are administered to a subject in an effective amount.

[0165] In some embodiments, the dose of viral vector administered to a subject will vary’ according to the age of the subject, the disease or disorder to be treated, and mode of administration. In some embodiments, the dose for achieving a therapeutic effect is a virus titer of at least about 102, 103, 104, 105, 106, 107, 108. 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016or more transducing units.

[0166] Administration of the pharmaceutically useful polynucleotide of the present disclosure or the polynucleotide cassette of the present disclosure is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility' of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington s ’ Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0167] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0168] A recombinant polynucleotide, plasmid, vector, and / or pharmaceutical composition of the present disclosure can be used in a method of treating a disorder in a subject in need thereof. A disorder can be a disease, a condition, a genotype, a phenotype, or any state associated with an adverse effect. In some embodiments, treating a disorder can comprise preventing, slowing progression of, reversing, or alleviating symptoms of the disorder. A method of treating a disorder can comprise delivering a recombinant polynucleotide as disclosed herein to a cell of a subject in need thereof and expressing the payload in the cell. A method of treating a disorder-90-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOcan comprise delivering a recombinant polynucleotide as disclosed herein to a cell of a subject in need thereof and expressing the payload in the cell. In some embodiments, a recombinant polynucleotide of the present disclosure can be used to treat a genetic disorder.

[0169] In some embodiments, the recombinant polynucleotides of the present disclosure express the payload, which results in increased protein expression levels corresponding to the payload in the cell, tissue, or subject. The recombinant polynucleotides of the present disclosure produce a from 1.1 -fold to 1000-fold increased protein expression in the cell, tissue, or subject. The recombinant polynucleotides of the present disclosure produce a from 1. 1-fold to 1000-fold, from 1.5-fold to 1000-fold, from 2-fold to 1000-fold, from 5-fold to 1000-fold, from 10-fold to 1000-fold, from 20-fold to 1000-fold, from 50-fold to 1000-fold, from 100-fold to 1000-fold, from 200-fold to 1000-fold, from 500-fold to 1000-fold, from 1.1-fold to 10-fold, from 1.5-fold to 10-fold, from 2-fold to 10-fold, from 5-fold to 10-fold, from 10-fold to 100-fold, from 20-fold to 100-fold, or from 50-fold to 100-fold increased protein expression in the cell, tissue, or subject. In some embodiments, the recombinant polynucleotides of the present disclosure produce at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold increased protein expression in the cell, tissue, or subject. Increase in protein expression can be measured by an assay comparing a sample or subject treated with the recombinant polynucleotide to a control sample or subject not treated with the recombinant polynucleotide.

[0170] In some embodiments, a recombinant polynucleotide expresses a protein in a first cell type e.g,. RPE cells that is not less than 0.001 -fold and not more than 100-fold, not less than 0.01-fold and not more than 100-fold, not less than 0.05-fold and not more than 100-fold, not less than 0. 1-fold and not more than 100-fold, not less than 0.2-fold and not more than 100-fold, not less than 0.25-fold and not more than 100-fold, not less than 0.5-fold and not more than 100- fold, not less than 1-fold and not more than 100-fold, not less than 5-fold and not less than 100- fold, not less than 10-fold and not more than 100-fold, not less than 0.001 -fold and not more than 50-fold, not less than 0.01-fold and not more than 50-fold, not less than 0.05-fold and not more than 50-fold, not less than 0. 1-fold and not more than 50-fold, not less than 0.2-fold and not more than 50-fold, not less than 0.25-fold and not more than 50-fold, not less than 0.5-fold and not more than 50-fold, not less than 1-fold and not more than 50-fold, not less than 5-fold and not more than 50-fold, not less than 10-fold and not more than 50-fold, not less than 0.001- fold and not more than 10-fold, not less than 0.01-fold and not more than 10-fold, not less than 0.05-fold and not more than 10-fold, not less than 0.1-fold and not more than 10-fold, not less than 0.2-fold and not more than 10-fold, not less than 0.25-fold and not more than 10-fold, not-91-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOless than 0.5-fold and not more than 10-fold, not less than 1-fold and not more than 10-fold, or not less than 5-fold and not more than 10-fold an expression level of the protein in a second cell ty pe, tissue ty pe, or organ.Diseases and Disorders

[0171] In some embodiments, a polynucleotide of the present disclosure or the recombinant polynucleotide cassette of the present disclosure may be administered using a viral vector (e.g., an AAV vector) to a subject in need thereof. In some embodiments, a polynucleotide comprising a 5’ UTR of the present disclosure or the recombinant polynucleotide cassette comprising a 5’ UTR of the present disclosure may be administered using a viral vector (e.g., an AAV vector) to a subject in need thereof. In some embodiments, a polynucleotide comprising a first 5’ UTR and a second 5’ UTR of the present disclosure or the recombinant polynucleotide cassette comprising a first 5’ UTR and a second 5’ UTR of the present disclosure may be administered using a viral vector (e.g.. an AAV vector) to a subject in need thereof. The payload may be selectively transcribed in the cell type or tissue type of interest, thereby preventing unwanted adverse effects in the subject due to expression in non-target tissues. The subject can be a human or a non-human animal. Thus, the polynucleotides disclosed herein, or the recombinant polynucleotide cassettes disclosed herein can serve as a therapeutically effective vector replacement therapy that senses endogenous nucleic acids to regulate expression of a transgene payload and prevent or minimize adverse side effects from overexpression of a transgene payload.

[0172] In some embodiments, the recombinant poly nucleotide of the disclosure or the recombinant polynucleotide cassette of the disclosure is used for treating a disease or disorder associated with abnormal expression of a gene or protein.

[0173] In some embodiments, a polynucleotide of the present disclosure or the recombinant polynucleotide cassette of the present disclosure may be administered using a viral vector (e.g., an AAV vector) to a subject in need thereof. Upon administration of said polynucleotide or said recombinant polynucleotide cassette, a payload sequence may’ be expressed in various target cell ty pes (e.g., RPE cells). The payload sequence may be selectively transcribed in the target cell ty pe, thereby preventing unwanted adverse effects in the subject due to expression in non-target tissues. The subject can be a human or a non-human animal. Thus, the polynucleotides disclosed herein, or the recombinant polynucleotide cassettes disclosed herein can serve as a therapeutically effective vector replacement therapy that senses endogenous nucleic acids to regulate expression of a transgene payload and prevent or minimize adverse side effects from overexpression of a transgene payload. This approach can be adapted for various cell ty pes and-92-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOtissues, depending on the specific therapeutic needs, by utilizing appropriate regulator}' elements and delivery strategies.

[0174] In some embodiments, the recombinant polynucleotides of the disclosure or the recombinant polynucleotide cassette of the disclosure can be used for treating an ocular disease. In some embodiments, the recombinant polynucleotides of the disclosure are used for treating a disease or disorder associated with the retinal pigment epithelium. Examples of retinal pigment epithelial diseases include but are not limited to age-related macular degeneration (AMD), Leber’s hereditary optic neuropathy, cone-rod dystrophy, Leber congenital amaurosis, Stargardt’s disease, diabetic retinopathy, retinal detachment, Best’s disease, retinitis pigmentosa, choroideremia and / or tapetoretinal degeneration.

[0175] In some embodiments, the targeted disease may be a disease which is not necessarily or specifically associated with the primary target cell type, but which can be treated or prevented by expressing specifically nucleic acid encoding a payload in that cell type. For example, in the case of ocular diseases, the target may be RPE cells, but this principle can be applied to other cell types and diseases in various organ systems.

[0176] In some embodiments, treatment of the targeted disease using the recombinant polynucleotides described herein may include their administration via appropriate routes for the specific tissue or organ system involved. For ocular diseases, this may include intraocular, subretinal or intravitreal administration. Similar tissue-specific administration routes can be employed for other target organs or tissues.

[0177] In some embodiments, the recombinant polynucleotides may be administered before or after the disease becomes symptomatic, e.g., before or after partial or complete degeneration of the target cell type and / or before or after partial or complete loss of the affected organ’s function. In the case of ocular diseases targeting RPE cells, this would refer to RPE degeneration and / or vision loss.Definitions

[0178] As used herein, the term "therapeutic polynucleotide” refers to a polynucleotide that is introduced into a cell and is capable of being expressed in the cell.

[0179] As used herein, the term “polynucleotide” refers to a single or double-stranded polymer of deoxy ribonucleotide (DNA) or ribonucleotide (RNA) bases read from the 5 ’ to the 3’ end.The term “RNA” is inclusive of dsRNA (double stranded RNA), snRNA (small nuclear RNA), IncRNA (long non-coding RNA), mRNA (messenger RNA), miRNA (microRNA) RNAi (inhibitory RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), tRNA (transfer-93-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WORNA), rRNA (ribosomal RNA), snoRNA (small nucleolar RNA), and cRNA (complementary RNA). The term DNA is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids.

[0180] The term “enhancer / promoter” refers to the combination of enhancer and core promoter sequences.

[0181] The term "‘ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0182] The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0183] Unless otherwise stated, whenever a range is recited, the range is inclusive of the recited endpoints.

[0184] The term percent “identity.” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0185] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows:[(Number of Identical Positions) / (Total Number of Positions in the Test Sequence)] x 100%-94-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0186] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words’") to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match / mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023, are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows:Percent Sequence Identity = (“Percent Identity” output value) x (“Query’ Coverage” output value)

[0187] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions ) / (total number of nucleotides in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 1 : AAAAAGGGGG (SEQ ID NO: 216) (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (SEQ ID NO: 217) (length = 10 nucleotides). The percent identity' between test sequence 1 and reference sequence 2 would be [(5) / (l 0)] x 100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 218) (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (SEQ ID NO: 219) (length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10) / (20)] xioo% = 50%.-95-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOTest sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (SEQ ID NO: 219) (length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 218) (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [( 10) / ( 10)] x 100% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6.

[0188] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions ) / (total number of amino acids in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (SEQ ID NO: 220) (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (SEQ ID NO: 221) (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5) / (l 0)] x ioo% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 222) (length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (SEQ ID NO: 220) (length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10) / (20)] *100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11 : FFFFFYYYYY (SEQ ID NO: 220) (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 222) (length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10) / (10)] xioo% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12.For purposes herein, reference to a polynucleotide sequence (e.g.. a DNA sequence or an RNA sequence) also encompasses the reverse complement of the polynucleotide sequence. For example, a sequence of AAAAAGGGGG (SEQ ID NO: 216) also encompasses a sequence of CCCCCTTTTT (SEQ ID NO: 223).

[0189] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g.. dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0190] As used herein, the term “effective amount” refers to the amount of a composition (e g., a synthetic peptide) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.-96-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0191] As used herein, the term '‘therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0192] As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g.. peptide) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g.. intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0193] As used herein, the term “antibody” is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”).

[0194] As used herein, the term “treatment” means an approach to obtaining a beneficial or intended clinical result. The beneficial or intended clinical result can include alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and / or improvement or alleviation of disease conditions.

[0195] As used herein, the term “pharmaceutical composition” refers to the combination of an active ingredient with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo.

[0196] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

[0197] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such as dimethylsulfoxide, N-methylpyrrolidone and mixtures thereof, and various types of wetting agents, solubilizing agents, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and-97-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOpreservatives. For examples of carriers, stabilizers and adjuvants, see. e.g., Martin, Remington ’s Pharmaceutical Sciences, 21st Ed., MackPubl. Co., Easton, Pa. (2005). incorporated herein by reference in its entirety.

[0198] As used herein, the singular forms “a,” ‘"an” and “the” include plural referents unless the context clearly dictates otherwise.

[0199] As used herein, the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).Embodiments

[0200] Embodiment 1. A recombinant polynucleotide comprising a 5’ untranslated region (5’ UTR) having a sequence with at least 80% sequence identity7to any one of SEQ ID NO: 1-9.

[0201] Embodiment 2. The recombinant polynucleotide of embodiment 1, wherein the sequence of the 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 1-9.

[0202] Embodiment 3. The recombinant polynucleotide of embodiment 2, wherein the sequence of the 5’ UTR is any one of SEQ ID NO: 1-9.

[0203] Embodiment 4. The recombinant polynucleotide of any one of embodiments 1-3, further comprising a payload operably linked to the 5?UTR.

[0204] Embodiment 5. The recombinant polynucleotide of embodiment 4, wherein the payload comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide.

[0205] Embodiment 6. The recombinant polynucleotide of embodiment 5, wherein the gene is MECP2, PRPH2, RHO, UBE3A, DYRK1A, MEF2C. NSD1. ATRX, RPS6KA3. TCF4, ZEB2, FOXG1, CDKL5, a partial piece of chromosome 2, SLC6A1, DMD, SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1 A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR. FLCN. FLCN, TP53, CRP, PTEN, IFT88. CLDN14, AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC1, BAP1, APOE, CYBA, GSTTL IFNG, IGFL IL2, ABCB1, SDHB, TSC2. BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB, NOX4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT, CYBA, CYBB, EGFR, HM0X1, LRP2, SERPINE1, PAX2, ABCB1, PPARA. PPARG, PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRASL CRB2, APRT, AXL, CCND1, CBR1, CPT1A. CYP1A1, CYP2B6, EDN1, ERBB2, HMGCR, MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2, PTGS2, HLTF, S0D1, SOD2, SREBF2, HNF1B, TERT, TNFSF10, NDRG1, MBTPS2, WNT4, BCOR, INF2, ALG9,-98-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOBICC1, TMEM67, IRX2, FREM1, ANKS6, FREM2, CD46, COL4A3, COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9.

[0206] Embodiment 7. The recombinant polynucleotide of any one of embodiments 4-6, further comprising a promoter operably coupled to the payload.

[0207] Embodiment 8. The recombinant polynucleotide of embodiment 7, wherein the promoter comprises a core promoter having a polynucleotide sequence of any one of SEQ ID NO: 49 - SEQ ID NO: 208.

[0208] Embodiment 9. The recombinant polynucleotide of any one of embodiments 4-8, wherein the recombinant polynucleotide, upon insertion into a cell, results in increased expression of the payload in the cell of from about 1.5-fold to about 9-fold, relative to an otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0209] Embodiment 10. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 2-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0210] Embodiment 11. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 3-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0211] Embodiment 12. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 4-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0212] Embodiment 13. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 5-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5‘ UTR.

[0213] Embodiment 14. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 6-fold, relative to the otherw ise comparable recombinant polynucleotide lacking the 5’ UTR.

[0214] Embodiment 15. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the-99-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOpayload in the cell of from about 7-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5 ’ UTR.

[0215] Embodiment 16. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the payload in the cell of from about 8-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0216] Embodiment 17. The recombinant polynucleotide of embodiment 9, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of the pay load in the cell of from about 9-fold, relative to the otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0217] Embodiment 18. The recombinant polynucleotide of any one of embodiments 9-17, wherein the cell is a neural cell, retinal cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell.

[0218] Embodiment 19. A recombinant polynucleotide comprising:(i) a 5’ untranslated region (5‘ UTR); and(ii) a pay load operably linked to the 5‘ UTR, wherein the recombinant polynucleotide, upon insertion into a retinal pigment epithelium (RPE) cell, results in increased expression of the payload in the RPE cell of at least 2-fold, relative to an otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

[0219] Embodiment 20. An engineered viral vector comprising the recombinant polynucleotide of any one of embodiments 1-19.

[0220] Embodiment 21. The engineered viral vector of embodiment 20, wherein the viral vector is an adeno-associated viral vector selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV-DJ.

[0221] Embodiment 22. A pharmaceutical composition comprising the recombinant polynucleotide of any one of embodiments 1-19 or the engineered viral vector of embodiment 20 or 21, and a pharmaceutically acceptable carrier.

[0222] Embodiment 23. A method of expressing a payload in a cell, the method comprising inserting into the cell a recombinant polynucleotide that comprises a 5' untranslated region (5’ UTR); and a pay load operably linked to the 5’ UTR, wherein the payload is expressed at a level that is from about 1.5-fold to about 9-fold higher than a control level of payload expression from inserting an otherwise comparable recombinant polynucleotide lacking the 5' UTR into the cell.-100-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

[0223] Embodiment 24. The method of embodiment 23, wherein the payload is expressed at a level that is about 2-fold higher than the control level of payload expression.

[0224] Embodiment 25. The method of embodiment 23, wherein the payload is expressed at a level that is about 3-fold higher than the control level of payload expression.

[0225] Embodiment 26. The method of embodiment 23, wherein the payload is expressed at a level that is about 4-fold higher than the control level of payload expression.

[0226] Embodiment 27. The method of embodiment 23, wherein the payload is expressed at a level that is about 5-fold higher than the control level of payload expression.

[0227] Embodiment 28. The method of embodiment 23, wherein the payload is expressed at a level that is about 6-fold higher than the control level of payload expression.

[0228] Embodiment 29. The method of embodiment 23, wherein the payload is expressed at a level that is about 7-fold higher than the control level of payload expression.

[0229] Embodiment 30. The method of embodiment 23, wherein the payload is expressed at a level that is about 8-fold higher than the control level of payload expression.

[0230] Embodiment 31. The method of embodiment 23, wherein the payload is expressed at a level that is about 9-fold higher than the control level of payload expression.

[0231] Embodiment 32. The method of any one of embodiments 23-31, wherein the cell is a neural cell, retinal cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell.

[0232] Embodiment 33. The method of embodiment 32, wherein the cell is a retinal cell.

[0233] Embodiment 34. The method of embodiment 33, wherein the retinal cell is a retinal pigment epithelium (RPE) cell.

[0234] Embodiment 35. The method of any one of embodiments 23-34, wherein 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 1-9.

[0235] Embodiment 36. The method of embodiment 35, wherein the sequence of the 5’ UTR is any one of SEQ ID NO: 1-9.

[0236] Embodiment 37. The method of any one of embodiments 23-36, wherein the payload comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide.

[0237] Embodiment 38. The method of embodiment 37, wherein the gene is MECP2, PRPH2, RHO, UBE3A. DYRK1A. MEF2C, NSD1, ATRX, RPS6KA3, TCF4, ZEB2. FOXG1, CDKL5, a partial piece of chromosome 2, SLC6A1, DMD, SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1 A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR, FLCN,-101-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOFLCN, TP53, CRP, PTEN, IFT88, CLDN14, AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC1, BAP1, APOE, CYBA, GSTT1, IFNG, IGF1, IL2, ABCB1, SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB, N0X4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT. CYBA, CYBB, EGFR, HM0X1, LRP2. SERPINE1, PAX2, ABCB1, PPARA. PPARG, PTGS2, RELA, RET, TLR4. UMOD, BAP1, RETN, GREB1L, FRAS1, CRB2, APRT, AXL, CCND1, CBR1, CPT1A, CYP1A1, CYP2B6, EDN1, ERBB2, HMGCR, MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2, PTGS2, HLTF, SOD1, SOD2, SREBF2, HNF1B, TERT, TNFSF1O, NDRG1, MBTPS2, WNT4, BCOR. INF2, ALG9, BICC1, TMEM67. IRX2, FREM1, ANKS6. FREM2, CD46, COL4A3. COL4A4, COL4A5. TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9.

[0238] Embodiment 39. A method of treating a disorder in a subject in need thereof, the method comprising administering to the subject the recombinant polynucleotide of any one of embodiments 1-19, the engineered viral vector of embodiment 20 or 21, or the pharmaceutical composition of embodiment 22, wherein the administering is sufficient to treat the disorder in the subj ect.

[0239] Embodiment 40. The method of embodiment 29, wherein the disorder is selected from the group consisting of age-related macular degeneration (AMD), Leber's hereditary optic neuropathy, cone-rod dystrophy. Leber congenital amaurosis, Stargardt's disease, diabetic retinopathy, retinal detachment, Best’s disease, retinitis pigmentosa, choroideremia, and tapetoretinal degeneration.EXAMPLES

[0240] The invention is further illustrated by the following non-limiting examples.EXAMPLE 15’ UTR Library Design and Screening

[0241] This example describes the screening of a 5' UTR library to identify sequences that enhance expression of a payload. A library of 10,000 5’ UTR sequences was compiled.

[0242] The 200 bp 5’ UTR sequences were cloned upstream of a GFP reporter gene in a lentiviral vector containing a BFP:P2A:Puromycin normalization cassette. HEK293T cells were transduced with the 5’ UTR lentiviral library'. After puromycin selection, BFP positive cells were sorted by FACS into bins based on GFP expression levels. The enrichment of 5’ UTR sequences in each GFP expression bin was then analyzed by NGS amplicon sequencing of genomic DNA. FIG. 1 shows an overview of this experimental workflow.-102-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOEXAMPLE 2Analysis of 5’ UTR Library Screen Results in HEK293T Cells

[0243] This example describes the analysis of results from the 5’ UTR library screen in HEK293T cells. Cells were sorted into four bins based on GFP expression levels: 0-40%, 50- 90%. 90-95%, and 95-100%. To determine the enrichment of 5’ UTR sequences in each bin. NGS analysis was performed on the sorted populations and compared to an unsorted cell population.

[0244] Initial results showed good correlation between biological replicates and distinct segregation between sorting bins (FIG. 2). As expected, negative control sequences with out-of- frame start codons were depleted in the high GFP expression bins and enriched in the low expression bins (FIG. 3).

[0245] Statistical analysis using DESeq2 identified several 5‘ UTR sequence candidates that were significantly enriched in the high GFP expression bins compared to the unsorted population (FIG. 4). However, further analysis revealed that these candidates did not necessarily show the expected pattern of depletion in the low GFP expression bins (FIG. 5). A complementary statistical testing approach used polyserial regression to identify 5’ UTR sequence candidates that displayed monotonically elevated abundance in sorted bins based on the percentile of the bin (e.g. greater abundance in 95-100% bin vs. 90-95% bin, greater abundance in 90-95% bin that 50-90% bin, etc.) (FIG. 6).EXAMPLE 3Analysis of 5’ UTR Library Screen Results in ARPE19 Cells

[0246] This example describes the analysis of a 5’ UTR library screen in ARPE19 cells. ARPE19 cells were sorted into four bins based on GFP expression levels: 0-40%, 50-90%, 90- 95%, and 95-100%. To determine the enrichment of 5’ UTR sequences in each bin, NGS data generation and analysis were carried out as performed for the analogous screen in HEK293T cells. 5’ UTR enrichments in high GFP bins versus unsorted bins were analyzed using two strategies: DESeq2- and polyserial regression-based testing. These methods were applied to both HEK293T and ARPE19 datasets, identifying additional and sometimes overlapping sets of hits. The analysis resulted in a combined set of 1590 5’ UTRs with statistical support by one or more methods in one or more methods in one or both cell types (FIG. 7).EXAMPLE 4Validation of 5’ UTR Hits Using a Dual Luciferase Reporter System

[0247] This example describes the validation of top 5’ UTR candidates from the HEK293T screen using a dual luciferase reporter system. Nine candidate 5' UTR sequences and-103-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOappropriate controls were cloned into an AAV vector containing a secreted Nanoluciferase (NLuc) reporter dow nstream of the 5’ UTR and a constitutively expressed Cypridina luciferase (CLuc) normalization gene (FIG. 8).

[0248] HEK293T cells were transduced with the AAV constructs with 10,000 viral genomes per cell. Luciferase activity was measured 2 days post-transduction. The top performing 5’ UTR sequences showed a 1 -9-fold increase in normalized NLuc activity compared to a no UTR control (FIG. 9). In FIG. 9, the order of bars in the graph is as follows: 1. Untransduced; 2. No UTR (Nluc translated); 3. No ATG (No start codon); 4. SEQ ID NO: 10; 5. SEQ ID NO: 1; 6. SEQ ID NO: 2; 7. SEQ ID NO: 3; 8. SEQ ID NO: 4; 9. SEQ ID NO: 5; 10. SEQ ID NO: 6; 11. SEQ ID NO: 7; 12. SEQ ID NO: 8; 13. SEQ ID NO: 9.

[0249] The most effective 5’ UTR sequences included SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. As expected, negative control sequences showed little to no NLuc activity. These negative controls included untransduced cells (first bar), no UTR (second bar), no ATG (third bar), and SEQ ID NO: 10 (fourth bar).

[0250] Similar results were observed when ARPE19 cells w ere transduced with the same viruses at 100,000 viral genomes per cell, with the top 5’ UTR hits showing comparable levels of translation enhancement (FIG. 10). In FIG. 10, the bar labeled “out of frame start’7corresponds to SEQ ID NO: 10; UTR 1 corresponds to SEQ ID NO: 1; UTR 2 corresponds to SEQ ID NO: 4; UTR 3 corresponds to SEQ ID NO: 9; UTR 4 corresponds to SEQ ID NO: 5; UTR 5 corresponds to SEQ ID NO: 7; UTR 6 corresponds to SEQ ID NO: 8; UTR 7 corresponds to SEQ ID NO: 3; UTR 8 corresponds to SEQ ID NO: 6; UTR 9 corresponds to SEQ ID NO: 2.

[0251] Top performing 5’ UTR sequences in ARPE-19 cells, UTR 8 (SEQ ID NO: 6) and UTR 9 (SEQ ID NO: 2), increase normalized NLuc activity 3.7 and 8.1 fold, respectively, compared to no 5’ UTR5’ UTR. Constructs containing UTR 8 (SEQ ID NO: 6) and UTR 9 (SEQ ID NO: 2) were also the top Nluc expressing constructs in iCell RPE cells (FIG. 11). In FIG. 11, the bar labeled “out of frame start” corresponds to SEQ ID NO: 10; UTR 1 corresponds to SEQ ID NO: 1; UTR 2 corresponds to SEQ ID NO: 4; UTR 3 corresponds to SEQ ID NO: 9; UTR 4 corresponds to SEQ ID NO: 5; UTR 5 corresponds to SEQ ID NO: 7; UTR 6 corresponds to SEQ ID NO: 8; UTR 7 corresponds to SEQ ID NO: 3; UTR 8 corresponds to SEQ ID NO: 6; UTR 9 corresponds to SEQ ID NO: 2.-104-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOEXAMPLE 5Testing of Additional 5’ UTRs by Transfection

[0252] This example describes testing additional 5’ UTR candidates using a dual luciferase reporter system. Thirteen candidate 5' UTR sequences and appropriate controls were cloned into an AAV vector containing a secreted Nanoluciferase (NLuc) reporter downstream of the 5’ UTR and a constitutively expressed Cypridina luciferase (CLuc) normalization gene.

[0253] ARPE19 cells and HEK cells were transfected with 500 ng of plasmid with the dual luciferase reporter system. Luciferase activity was measured 2 days post-transfection. In FIG. 12 and FIG. 13, the order of bars in the graph is as follows: 1. Untransfected; 2. No ATG (No start codon); 3. SEQ ID NO: 10; 4. No UTR (Nluc translated); 5. SEQ ID NO: 12; 6. SEQ ID NO: 13; 7. SEQ ID NO: 14; 8. SEQ ID NO: 15; 9. SEQ ID NO: 16; 10. SEQ ID NO: 17; 11. SEQ ID NO: 18; 12. SEQ ID NO: 19; 13. SEQ ID NO: 20; 14. SEQ ID NO: 21; 15. SEQ ID NO: 22; 16. SEQ ID NO: 23; 17. SEQ ID NO: 24; 18. SEQ ID NO: 6; 19. SEQ ID NO: 2.

[0254] Based on consistent performance over the no UTR control in both cell lines, three 5’ UTR sequences showed an increase in normalized NLuc activity7compared to a no UTR control (i.e., “Fold-change” in FIG. 12 and FIG. 13) - SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 24 (bars 11, 14, and 17, respectively). As expected, negative control sequences showed little to no NLuc activity. These negative controls included untransduced cells (first bar), no ATG (second bar), SEQ ID NO: 10 (third bar), and no UTR (fourth bar).

[0255] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.-105-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A recombinant polynucleotide comprising a first 5’ untranslated region (5’ UTR) having a sequence with at least 80% sequence identity to any one of SEQ ID NO:

2. SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

2. The recombinant polynucleotide of claim 1, wherein the sequence of the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-243. The recombinant polynucleotide of claim 1 or claim 2, wherein the sequence of the first 5?UTR is any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9. SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

4. The recombinant polynucleotide of any one of claims 1-3. wherein the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

5. The recombinant polynucleotide of any one of claims 1-3, wherein the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

6. The recombinant polynucleotide of any one of claims 1-4, wherein the first 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

7. The recombinant polynucleotide of any one of claims 1-3 or 5, wherein the first 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21.

8. The recombinant polynucleotide of any one of claims 1-4 or 6, wherein the first 5’ UTR has a sequence of SEQ ID NO: 2.

9. The recombinant polynucleotide of any one of claims 1-3, 5, or 7, wherein the first 5’ UTR has a sequence of SEQ ID NO: 21.

10. The recombinant polynucleotide of any one of claims 1-9, further comprising a first payload operably linked to the first 5’ UTR.

11. The recombinant polynucleotide of any one of claims 1-10, further comprising a second 5’ UTR having a sequence with at least 80% sequence identity to any one of SEQ ID NO: 21,-106-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOSEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24, wherein the first and second 5’ UTRs are different.

12. The recombinant polynucleotide of claim 11, wherein the sequence of the second5?UTR has at least 90% sequence identity to any one of SEQ ID NO: 21, SEQ ID NO: 2; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

13. The recombinant polynucleotide of claim 11 or claim 12, wherein the sequence of the second 5‘ UTR is any one of SEQ ID NO:

21. SEQ ID NO: 2; SEQ ID NO: 1, SEQ ID NO: 3-9. SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

14. The recombinant polynucleotide of any one of claims 10-13, wherein the sequence of the second 5?UTR has at least 80% sequence identity to SEQ ID NO: 2.

15. The recombinant polynucleotide of any one of claims 10-13, wherein the sequence of the second 5’ UTR has at least 80% sequence identity to SEQ ID NO: 21.

16. The recombinant polynucleotide of any one of claims 10-14, wherein the sequence of the second 5’ UTR has at least 90% sequence identity' to SEQ ID NO: 2.

17. The recombinant polynucleotide of any one of claims 10-13 or 15, wherein the sequence of the second 5’ UTR has at least 90% sequence identity to SEQ ID NO: 21.

18. The recombinant polynucleotide of any one of claims 10-14 or 16, wherein the sequence of the second 5’ UTR is SEQ ID NO: 2.

19. The recombinant polynucleotide of any one of claims 10-13, 15, or 17, wherein the sequence of the second 5’ UTR is SEQ ID NO: 21.

20. The recombinant polynucleotide of any one of claims 11-19, wherein the first 5’ UTR is any one of SEQ ID NO: 2, SEQ ID NO: 1, and SEQ ID NO: 3-9, and the second 5' UTR is any one of SEQ ID NO: 21, SEQ ID NO: 12-20 and SEQ ID NO: 22-24.

21. The recombinant polynucleotide of any one of claims 11-19, wherein the first 5’ UTR is SEQ ID NO: 2 and the second 5’ UTR is SEQ ID NO: 21.

22. The recombinant polynucleotide of any one of claims 1-3 and 10-20, wherein the first 5’ UTR is SEQ ID NO: 2 or SEQ ID NO: 21-107-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO23. The recombinant polynucleotide of any one of claims 1-13, wherein the second 5’ UTR is SEQ ID NO: 2 or SEQ ID NO: 21.

24. The recombinant polynucleotide of any one of claims 10-23, further comprising a second payload operably linked to the second 5‘ UTR.

25. The recombinant polynucleotide of any one of claims 10-24, wherein the first payload and / or the second payload each comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide.

26. The recombinant polynucleotide of claim 25, wherein the gene is MECP2, PRPH2, RHO, UBE3A. DYRK1A, MEF2C, NSD1, ATRX, RPS6KA3, TCF4, ZEB2, FOXG1. CDKL5, a partial piece of chromosome 2. SLC6A1, DMD. SERPINA1, ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1, IL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFB1, UMOD, BCOR, FLCN, FLCN, TP53, CRP, PTEN, IFT88, CLDN14, AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC1, BAPL APOE. CYBA, GSTT1, IFNG, IGF1, IL2, ABCBL SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRGL GANAB, N0X4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT, CYBA, CYBB, EGFR, HM0X1, LRP2, SERPINE1, PAX2, ABCB1, PPARA, PPARG, PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRAS1, CRB2, APRT, AXL. CCND1, CBR1, CPT1A, CYP1A1, CYP2B6. EDNL ERBB2, HMGCR. MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2. PTGS2, HLTF, SODL SOD2, SREBF2, HNF1B, TERT, TNFSF10, NDRGL MBTPS2, WNT4, BCOR, INF2, ALG9, BICC1, TMEM67, IRX2, FREM1, ANKS6, FREM2, CD46, COL4A3, COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9.

27. The recombinant polynucleotide of any one of claims 24-26, further comprising (i) a first promoter operably coupled to both the first and second payloads, or (ii) a first promoter operably coupled to the first payload and a second promoter operably coupled to the second payload.

28. The recombinant polynucleotide of claim 27, wherein each of the first and / or second promoter comprises a core promoter having a polynucleotide sequence of any one of SEQ ID NO: 49 - SEQ ID NO: 208.

29. The recombinant polynucleotide of claim 28, wherein the first promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 52.-108-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO30. The recombinant polynucleotide of claim 28, wherein the first promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 53.

31. The recombinant polynucleotide of claim 28 or 30, wherein the second promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 52.

32. The recombinant polynucleotide of claim 28 or 29, wherein the second promoter comprises a core promoter having a polynucleotide sequence of SEQ ID NO: 53.

33. The recombinant polynucleotide of any one of claims 24-32, wherein the recombinant polynucleotide, upon insertion into a cell, results in increased expression of each of the first payload and / or the second pay load in the cell of from about 1.5-fold to about 9-fold, relative to an otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

34. The recombinant polynucleotide of claim 33, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first payload and / or the second pay load in the cell of from about 2-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

35. The recombinant polynucleotide of claim 34, wherein the recombinant poly nucleotide, upon insertion into the cell, results in increased expression of each of the first pay load and / or the second payload in the cell of from about 3-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

36. The recombinant polynucleotide of claim 35, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first pay load and / or the second pay load in the cell of from about 4-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

37. The recombinant polynucleotide of claim 36, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first pay load and / or the second pay load in the cell of from about 5-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5' UTR.

38. The recombinant polynucleotide of claim 37, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first payload and / or the second pay load in the cell of from about 6-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.-109-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO39. The recombinant polynucleotide of claim 38, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first pay load and / or the second payload in the cell of from about 7-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

40. The recombinant polynucleotide of claim 39, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first pay load and / or the second pay load in the cell of from about 8-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

41. The recombinant polynucleotide of claim 40, wherein the recombinant polynucleotide, upon insertion into the cell, results in increased expression of each of the first payload and / or the second pay load in the cell of from about 9-fold, relative to the otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

42. The recombinant polynucleotide of any one of claims 33-41, wherein the cell is a neural cell, retinal cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell.

43. The recombinant polynucleotide of any one of claims 1-42 further comprising a WPRE.

44. The recombinant polynucleotide of any one of claims 43, wherein the WPRE comprises at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 214.

45. The recombinant polynucleotide of any one of claims 1-44 further comprising a polyA sequence.

46. The recombinant polynucleotide of any one of claims 45, wherein the polyA is a bovine growth hormone polyA sequence.

47. The recombinant polynucleotide of any one of claims 45, wherein the polyA sequence comprises at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 215.

48. A recombinant polynucleotide comprising:(i) a 5’ untranslated region (5’ UTR); and-110-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO(ii) a pay load operably linked to the 5’ UTR, wherein the recombinant polynucleotide, upon insertion into a retinal pigment epithelium (RPE) cell, results in increased expression of the pay load in the RPE cell of at least 2-fold, relative to an otherwise comparable recombinant polynucleotide lacking the 5’ UTR.

49. The recombinant polynucleotide of claim 48, wherein the 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

50. The recombinant polynucleotide of claim 48 or claim 49, wherein the 5’ UTR has a sequence with at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

51. The recombinant polynucleotide of any one of claims 48-50, wherein the 5’ UTR has a sequence of any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

52. The recombinant polynucleotide of any one of claims 48-51, wherein the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

53. The recombinant polynucleotide of any one of claims 48-51, wherein the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

54. The recombinant polynucleotide of any one of claims 48-52, wherein the 5‘ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

55. The recombinant polynucleotide of any one of claims 48-51 or 53, wherein the 5‘ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21.

56. The recombinant polynucleotide of any one of claims 48-52 or 54, wherein the 5’ UTR has a sequence of SEQ ID NO: 2.

57. The recombinant polynucleotide of any one of claims 48-51, 53, or 55, wherein the 5’ UTR has a sequence of SEQ ID NO: 21.

58. A recombinant polynucleotide comprising:(i) a first 5' untranslated region (5‘ UTR),(ii) a first payload operably linked to the first 5’ UTR,-111-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO(iii) a second 5’ UTR, and(iv) a second payload operably linked to the second 5 ’ UTR; and wherein the recombinant polynucleotide, upon insertion into a retinal pigment epithelium (RPE) cell, results in increased expression of the first payload and / or second payload in the RPE cell of at least 2-fold, relative to an otherwise comparable recombinant polynucleotide lacking the corresponding 5’ UTR.

59. The recombinant polynucleotide of claim 58, wherein the sequence of the first 5' UTR has at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1 , SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

60. The recombinant polynucleotide of claim 58 or claim 59, wherein the sequence of the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO:

2. SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

61. The recombinant polynucleotide of any one of claims 58-60, wherein the sequence of the first 5’ UTR is any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO:

1. SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

62. The recombinant polynucleotide of any one of claims 58-61, wherein the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

63. The recombinant polynucleotide of any one of claims 58-61, wherein the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

64. The recombinant polynucleotide of any one of claims 58-62, wherein the first 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

65. The recombinant polynucleotide of any one of claims 58-61 or 63, wherein the first 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21.

66. The recombinant polynucleotide of any one of claims 58-62 or 64, wherein the first 5’ UTR has a sequence of SEQ ID NO: 2.

67. The recombinant polynucleotide of any one of claims 58-61, 63, or 65, wherein the first 5’ UTR has a sequence of SEQ ID NO: 21.-112-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO68. The recombinant polynucleotide of any one of claims 58-67, wherein the sequence of the second 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

69. The recombinant polynucleotide of any one of claims 58-68, wherein the sequence of the second 5’ UTR has at least 90% sequence identity' to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

70. The recombinant polynucleotide of any one of claims 58-69, wherein the sequence of the second 5' UTR is any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

71. The recombinant polynucleotide of any one of claims 58-70, wherein the second 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

72. The recombinant polynucleotide of any one of claims 58-70, wherein the second 5" UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

73. The recombinant polynucleotide of any one of claims 58-71, wherein the second 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

74. The recombinant polynucleotide of any one of claims 58-70 or 72, wherein the second 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21.

75. The recombinant polynucleotide of any one of claims 58-71 or 73, wherein the second 5’ UTR has a sequence of SEQ ID NO: 2.

76. The recombinant polynucleotide of any one of claims 58-70, 72, or 74, wherein the second 5‘ UTR has a sequence of SEQ ID NO: 21.

77. The recombinant polynucleotide of any one of claims 58-76, wherein the first 5’ UTR is SEQ ID NO: 2 and the second 5’ UTR is SEQ ID NO: 21.

78. An engineered viral vector comprising the recombinant polynucleotide of any one of claims 1-77.

79. The engineered viral vector of claim 78. wherein the viral vector is an adeno-associated viral vector selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV-DJ.-113-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO80. A pharmaceutical composition comprising the recombinant polynucleotide of any one of claims 1-77 or the engineered viral vector of claim 78 or 79, and a pharmaceutically acceptable carrier.

81. A method of expressing a payload in a cell, the method comprising inserting into the cell a recombinant polynucleotide that comprises a 5’ untranslated region (5’ UTR); and a payload operably linked to the 5’ UTR, wherein the payload is expressed at a level that is from about 1.5-fold to about 9-fold higher than a control level of payload expression from inserting an otherwise comparable recombinant polynucleotide lacking the 5’ UTR into the cell.

82. The method of claim 81, wherein the payload is expressed at a level that is about 2-fold higher than the control level of payload expression.

83. The method of claim 81, wherein the payload is expressed at a level that is about 3-fold higher than the control level of payload expression.

84. The method of claim 81. wherein the payload is expressed at a level that is about 4-fold higher than the control level of payload expression.

85. The method of claim 81, wherein the payload is expressed at a level that is about 5-fold higher than the control level of payload expression.

86. The method of claim 81, wherein the payload is expressed at a level that is about 6-fold higher than the control level of payload expression.

87. The method of claim 81 , wherein the payload is expressed at a level that is about 7-fold higher than the control level of payload expression.

88. The method of claim 81. wherein the payload is expressed at a level that is about 8-fold higher than the control level of payload expression.

89. The method of claim 81, wherein the payload is expressed at a level that is about 9-fold higher than the control level of payload expression.

90. The method of any one of claims 81-89, wherein the 5’ UTR has a sequence with at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.-114-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO91. The method of any one of claims 81 -90, wherein the 5 ’ UTR has a sequence with at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

92. The method of any one of claims 81-91, wherein the 5’ UTR has a sequence of any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

93. The method of any one of claims 81-92. wherein the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

94. The method of any one of claims 81-92, wherein the 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

95. The method of any one of claims 81-93, wherein the 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

96. The method of any one of claims 81-92 or 94, wherein the 5’ UTR has a sequence with at least 90% sequence identity7to SEQ ID NO: 21.

97. The method of any one of claims 81-93 or 95. wherein the 5’ UTR has a sequence of SEQ ID NO: 2.

98. The method of any one of claims 81-92, 94, or 96. wherein the 5' UTR has a sequence of SEQ ID NO: 21.

99. A method of expressing a first payload and a second payload in a cell, the method comprising inserting into the cell a recombinant polynucleotide that comprises:(i) a first 5’ untranslated region (5’ UTR),(ii) a first payload operably linked to the first 5’ UTR,(iii) a second 5’ UTR, and(iv) a second payload operably linked to the second 5 ’ UTR; and wherein the first pay load and / or the second pay load is each expressed at a level that is from about 1.5-fold to about 9-fold higher than a first control level of the first payload expression and / or a second control level of the second payload expression, wherein the fold change is from inserting an otherwise comparable recombinant polynucleotide lacking the corresponding 5' UTR into the cell.-115-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO100. The method of claim 99, wherein the first payload and / or the second payload is expressed at a level that is about 2-fold higher than the first control level of the first pay load expression and / or the second control level of the second pay load expression.

101. The method of claim 99, wherein the first payload and / or the second payload is expressed at a level that is about 3-fold higher than the first control level of the first payload expression and / or the second control level of the second pay load expression.

102. The method of claim 99. wherein the first payload and / or the second payload is expressed at a level that is about 4-fold higher than the first control level of the first pay load expression and / or the second control level of the second pay load expression.

103. The method of claim 99. wherein the first payload and / or the second payload is expressed at a level that is about 5-fold higher than the first control level of the first payload expression and / or the second control level of the second pay load expression.

104. The method of claim 99. wherein the first payload and / or the second payload is expressed at a level that is about 6-fold higher than the first control level of the first pay load expression and / or the second control level of the second pay load expression.

105. The method of claim 99. wherein the first payload and / or the second payload is expressed at a level that is about 7-fold higher than the first control level of the first pay load expression and / or the second control level of the second pay load expression.

106. The method of claim 99, wherein the first payload and / or the second payload is expressed at a level that is about 8-fold higher than the first control level of the first pay load expression and / or the second control level of the second pay load expression.

107. The method of claim 99, wherein the first payload and / or the second payload is expressed at a level that is about 9-fold higher than the first control level of the first pay load expression and / or the second control level of the second pay load expression.

108. The method of any one of claims 99-107, wherein the sequence of the first 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.-116-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO109. The method of any one of claims 99-108, wherein the sequence of the first 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

110. The method of any one of claims 99-109, wherein the sequence of the first 5’ UTR is any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

111. The method of any one of claims 99-110, wherein the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

112. The method of any one of claims 99-110, wherein the first 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

113. The method of any one of claims 99-111, wherein the first 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

114. The method of any one of claims 99-110 or 112, wherein the first 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21.

115. The method of any one of claims 99-111 or 113. wherein the first 5?UTR has a sequence of SEQ ID NO: 2.

116. The method of any one of claims 99-110, 112. or 1114, wherein the first 5‘ UTR has a sequence of SEQ ID NO: 21.

117. The method of any one of claims 99-116, wherein the sequence of the second 5’ UTR has at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9. SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

118. The method of any one of claims 99-117, wherein the sequence of the second 5’ UTR has at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9. SEQ ID NO: 12-20, and SEQ ID NO: 22-24.

119. The method of any one of claims 99-118, wherein the sequence of the second 5’ UTR is any one of SEQ ID NO: 2, SEQ ID NO: 21; SEQ ID NO: 1, SEQ ID NO: 3-9, SEQ ID NO: 12- 20, and SEQ ID NO: 22-24.-117-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO120. The method of any one of claims 99-119, wherein the second 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 2.

121. The method of any one of claims 99-119, wherein the second 5’ UTR has a sequence with at least 80% sequence identity to SEQ ID NO: 21.

122. The method of any one of claims 99-120, wherein the second 5' UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 2.

123. The method of any one of claims 99-119 or 121, wherein the second 5’ UTR has a sequence with at least 90% sequence identity to SEQ ID NO: 21.

124. The method of any one of claims 99-120 or 122, wherein the second 5’ UTR has a sequence of SEQ ID NO: 2.

125. The method of any one of claims 99-119, 121. or 123. wherein the second 5’ UTR has a sequence of SEQ ID NO: 21.

126. The method of any one of claims 99-125, wherein the first 5’ UTR is SEQ ID NO: 2 and the second 5’ UTR is SEQ ID NO: 21.

127. The method of any one of claims 81-126, wherein the cell is a neural cell, retinal cell, lung cell, epithelial cell, skeletal muscle cell, dendritic cell, hepatic cell, pancreatic cell, bone cell, hematopoietic stem cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, or heart cell.

128. The method of any one of claims 81-127, wherein the cell is a retinal cell.

129. The method of claim 128, wherein the retinal cell is a retinal pigment epithelium (RPE) cell.

130. The method of any one of claims 81-129, wherein the payload, the first payload, the second payload, or any combination thereof, comprises a gene that encodes a therapeutic protein or a therapeutic polynucleotide.

131. The method of claim 130, wherein the gene is MECP2, PRPH2. RHO, UBE3A, DYRK1A, MEF2C, NSD1, ATRX, RPS6KA3, TCF4, ZEB2. FOXG1. CDKL5, a partial piece of chromosome 2, SLC6A1 , DMD, SERPINA1 , ABCA4, CFTR, HEXA, RAB7A, ATP7B, HFE, LIPA, SCNN1A, PKD1, PKD2, PKHD1, ACE, ALB, VHL, EPO, FH, ACE, TNF, SPP1,-118-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WOIL6, MYH9, TSC2, ADIPOQ, IL2, CCL2, TGFBL UMOD, BCOR, FLCN, FLCN, TP53, CRP, PTEN, IFT88, CLDN14, AGT, MET, MYH9, YWHAE, HAMP, EPO, MUC1, BAP1, APOE, CYBA, GSTT1, IFNG, IGF1, IL2, ABCB1, SDHB, TSC2, BRAF, CDKN1B, GLA, KRT7, PPARG, RET, TRPC6, NDRG1, GANAB, N0X4, ADIPOR1, GREB1L, ANKS6, NUTM2B, CAT, CYBA, CYBB, EGFR, HM0X1, LRP2. SERPINE1, PAX2, ABCB1, PPARA. PPARG. PTGS2, RELA, RET, TLR4, UMOD, BAP1, RETN, GREB1L, FRAS1, CRB2, APRT, AXL, CCND1, CBR1, CPT1A, CYP1A1, CYP2B6, EDN1, ERBB2, HMGCR, MME, NFKB1, NGF, MAPK1, MAPK3, PTGS2, PTGS2, HLTF, S0D1, SOD2, SREBF2, HNF1B, TERT, TNFSF10, NDRG1, MBTPS2, WNT4, BCOR, INF2, ALG9. BICC1, TMEM67, IRX2, FREM1, ANKS6, FREM2, CD46, COL4A3, COL4A4, COL4A5, TTC21B, NPHP4, CD2AP, CFI, LAMB2, LMX1B, or MYH9.

132. A method of treating a disorder in a subject in need thereof, the method comprising administering to the subject the recombinant polynucleotide of any one of claims 1-77, the engineered viral vector of claim 78 or 79, or the pharmaceutical composition of claim 80, thereby treating the disorder in the subject.

133. The method of claim 132, wherein the disorder is selected from the group consisting of age-related macular degeneration (AMD), Leber’s hereditary optic neuropathy, cone-rod dystrophy, Leber congenital amaurosis, Stargardt's disease, diabetic retinopathy, retinal detachment, Best’s disease, retinitis pigmentosa, choroideremia, and tapetoretinal degeneration.-119-KT S Ref. : 116779- 1521686-740WO 1ShapeTX Ref. No.: STX-130WO

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