WO2024031060A1

WO2024031060A1 - Lentiviral vectors and uses thereof

Info

Publication number: WO2024031060A1
Application number: PCT/US2023/071690
Authority: WO
Inventors: Luis AGOSTO; Robert Blakemore; Christopher Driscoll; Stacie SEIDEL
Original assignee: Elevatebio Technologies, Inc.
Priority date: 2022-08-04
Filing date: 2023-08-04
Publication date: 2024-02-08

Abstract

The present disclosure provides lentiviral transfer vectors comprising a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G and a truncated 5' long terminal repeat (Δ5' LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four nucleotides at the 5' end of the Δ 5' LTR consist of GGGX, wherein X is A, T, or C. The present disclosure further provides packaging vectors, systems and kits comprising the transfer and packaging vectors, and methods of producing lentiviral particles.

Description

LENTIVIRAL VECTORS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/395,016, filed August 4, 2022, the contents of which are incorporated herein by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The contents of the text file submitted electronically herewith are incorporated by reference in their entirety: a computer readable format copy of the Sequence Listing (filename: ELVT_014_01WO_SeqList_ST26.xml, size 158KB, generated July 31 , 2023).

BACKGROUND

[0003] Viral vectors are efficient gene delivery tools in eukaryotic cells. Nayerossadat. Adv Biomed Res. 2012; 1 : 27. Useful viral vectors have been created from different virus families, including retroviruses such as lentiviruses. Nayerossadat. Adv Biomed Res. 2012; 1 : 27. Lentiviruses have proven to be versatile and effective gene transfer vectors for a variety of applications since they are easy to manipulate, typically do not induce a strong anti-viral immune response and are able to integrate into the genome of a host cell, leading to stable gene expression. Nayerossadat. Adv Biomed Res. 2012; 1 : 27. If provided with an appropriate envelope protein, lentiviruses can infect almost any type of cell. Dautzenberg. Gene Therapy. 2021 ; 28: 89-104. Due to these advantages, a large number of lentiviral vectors have been developed for in vitro gene transfer. In addition, use of lentiviruses for purposes such as the creation of transgenic or knockout animals, or for gene therapy, has been explored. However, there remains a need in the art for improved lentiviral systems, e.g., with higher crude titer, lower particle to infectivity (P:l) ratio, or both.

SUMMARY

[0004] Provided herein is a lentiviral transfer vector comprising: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four nucleotides at the 5’ end of the A 5’ LTR consist of GGGX, wherein X is A, T, or C; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

[0005] In embodiments, the lentiviral transfer vector further comprises: a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence comprising a truncated Gag gene (AGag); a polynucleotide sequence comprising a truncated Envelope gene (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; and a polypurine tract (PPT) polynucleotide sequence.

[0006] In embodiments, the A5’ LTR of a lentiviral transfer vector described herein comprises an R region and a U5 region of an HIV 5’ LTR. In embodiments, the A5’ LTR of a lentiviral transfer vector described herein consists of an R region and a U5 region of an HIV 5’ LTR. In embodiments, the HIV 5’ LTR is from HIV-1 subtype B. In embodiments, the 3’ LTR is a self-inactivating 3’ LTR (SIN 3’ LTR). In embodiments, the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR. In embodiments, the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR. In embodiments, the HIV 3’LTR is from HIV-1 subtype B. In embodiments, the A 5’ LTR and 3’ LTR are from the same HIV subtype. In embodiments, pA of a lentiviral transfer vector described herein is a bovine growth hormone pA (bGH-pA). In embodiments, the location for inserting the expression cassette in the lentiviral transfer vector is a multiple cloning site (MCS).

[0007] In embodiments, provided herein is a lentiviral transfer vector, comprising, from 5’ to 3’: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv) wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

[0008] In embodiments, provided herein is a lentiviral transfer vector comprising a splice donor site and a splice acceptor site. In embodiments, the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence. In embodiments, the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence. In embodiments, the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR. In embodiments, the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR. In embodiments, the HIV is HIV-1 subtype B. In embodiments, the 3’ LTR of the lentiviral transfer vector is a self-inactivating 3’ LTR (SIN 3’ LTR). In embodiments, the SIN 3’ LTR of the lentiviral transfer vector comprises an R region and a U5 region of an HIV 3’ LTR. In embodiments, the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR. In embodiments, the HIV is HIV-1 subtype B. In embodiments, the A 5’ LTR and 3’ LTR are from the same HIV subtype. In embodiments, the pA is a bovine growth hormone pA (bGH-pA). In embodiments, the location for inserting the expression cassette is a multiple cloning site (MCS). In embodiments, the CMV promoter polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 1 . In embodiments, the A5’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:2 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 2. In embodiments, the 3’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 3. In embodiments, the pA polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:4 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 4. In embodiments, the Psi ( ¹) viral packaging signal polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:5 or a nucleic acid with at least about 95 %, at least about 96 %, at least about

97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 5. In embodiments, the AGag polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:6 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about

98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 6. In embodiments, the AEnv polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:7 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 7. In embodiments, the central polypurine tract (cPPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:8 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 8. In embodiments, the polypurine tract (PPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:9 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 9. In embodiments, the lentiviral transfer vector comprises the polynucleotide sequence of SEQ ID NO:10 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 10. In embodiments, the AGag-AEnv-RRE of a lentiviral transfer vector comprises the polynucleotide sequence of SEQ ID NO: 15 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 15. In embodiments, the AGag-AEnv-RRE of a lentiviral transfer vector comprises the polynucleotide sequence of SEQ ID NO: 17 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 17. In embodiments, the AGag-AEnv-RRE of a lentiviral transfer vector comprises the polynucleotide sequence of SEQ ID NO: 19 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 19. In embodiments, provided herein is a lentiviral packaging vector comprising, from 5’ to 3’: (i) an RSV promoter polynucleotide sequence; (ii) a polynucleotide sequence encoding a Rev protein; and (iii) an HSV thymidine kinase polyadenylation polynucleotide sequence. In embodiments, the lentiviral packaging vector comprises the polynucleotide sequence of SEQ ID NO:11 ; or a sequence with at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity thereto.

[0009] In embodiments, provided herein is a lentiviral packaging vector comprising, from 5’ to 3’: (i) a CMV promoter polynucleotide sequence; (ii) a human p-globin intron polynucleotide sequence; (iii) a polynucleotide sequence encoding a Gag protein, a Pol protein, and a Rev response element (RRE); and (iv) a rabbit p-globin polyadenylation polynucleotide sequence. In embodiments, the lentiviral packaging vector comprises the polynucleotide sequence of SEQ ID NO: 12; or a sequence with at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity thereto.

[0010] In embodiments, provided herein is a lentiviral packaging vector comprising, from 5’ to 3’: (i) a CMV promoter polynucleotide sequence; (ii) a rabbit p-globin intron polynucleotide sequence; (iii) a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and (iv) a human p-globin polyadenylation polynucleotide sequence. In embodiments, the lentiviral packaging vector comprises the polynucleotide sequence of SEQ ID NO:13; or a sequence with at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identity thereto.

[0011] In embodiments, provided herein is a lentiviral vector system comprising: (a) a first packaging vector comprising a polynucleotide sequence encoding a Rev protein; (b) a second packaging vector comprising a polynucleotide sequence encoding a Gag protein and a Pol protein; (c) a third packaging vector comprising a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and (d) a transfer vector comprising, from 5’ to 3’: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence. In embodiments, the transfer vector further comprises a splice donor site and a splice acceptor site. In embodiments, the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence. In embodiments, the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence. In embodiments, the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR. In embodiments, the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR. In embodiments, the HIV of the HIV 5’ LTR is HIV-1 subtype B. In embodiments, the 3’ LTR is a self-inactivating 3’ LTR (SIN 3’ LTR). In embodiments, the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR. In embodiments, the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR. In embodiments, the HIV is HIV-1 subtype B. In embodiments, the A 5’ LTR and 3’ LTR are from the same HIV subtype. In embodiments, the pA is a bovine growth hormone pA (bGH-pA). In embodiments, the location for inserting the expression cassette is a multiple cloning site (MCS). In embodiments, the CMV promoter polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:1. In embodiments, the A5’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:2. In embodiments, the 3’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:3. In embodiments, the pA polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:4. In embodiments, the Psi (T) viral packaging signal polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:5. In embodiments, the AGag polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:6. In embodiments, the AEnv polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:7. In embodiments, the central polypurine tract (cPPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:8. In embodiments, the polypurine tract (PPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:9. In embodiments, the transfer vector comprises the polynucleotide sequence of SEQ ID NO:10. In embodiments, the first packaging vector comprises a polynucleotide sequence of SEQ ID NO:1 1 . In embodiments, the second packaging vector comprises a polynucleotide sequence of SEQ ID NO:12. In embodiments, the third packaging vector comprises a polynucleotide sequence of SEQ ID NO:13.

[0012] Provided herein is a lentiviral vector system, comprising: (i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:1 1 ; (iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13. In embodiments, the lentiviral vector system is able to generate a crude titer of at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL. In embodiments, the lentiviral vector system is able to generate a crude titer of at least 1 E8 TU/mL. In embodiments, the lentiviral vector system is able to generate a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200. In embodiments, the lentiviral vector system is able to generate a P:l of at most 200. In embodiments, the lentiviral vector system comprises an expression cassette comprising a polynucleotide encoding the GOI. In embodiments, the expression cassette comprises an EF1a promoter polynucleotide sequence. In embodiments, the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide. In embodiments, the woodchuck hepatitis virus post-transcriptional regulatory element is a woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. In embodiments, the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39. In embodiments, the expression cassette comprises from 5’ to 3’: (i) an EF1a promoter polynucleotide sequence; (ii) a polynucleotide encoding the GOI; and (iii) woodchuck hepatitis virus post-transcriptional regulatory element (oPRE) variant 1 or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. In embodiments, the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system. In embodiments, the lentiviral vector system further comprises an expression cassette comprising a polynucleotide encoding the GOI. In embodiments, the expression cassette comprises an EF1a promoter polynucleotide sequence. In embodiments, the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide. In embodiments, the woodchuck hepatitis virus post-transcriptional regulatory element is a woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. In embodiments, the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39. In embodiments, the expression cassette comprises from 5’ to 3’: (i) an EF1a promoter polynucleotide sequence;(ii) a polynucleotide encoding the GOI; and (iii) woodchuck hepatitis virus post-transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. In embodiments, the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

[0013] In embodiments, provided herein is a method of producing a lentivirus, comprising: (i) introducing the lentiviral vector system described herein into a cell; (ii) culturing the cell under conditions where a lentivirus is produced; and (iii) harvesting the lentivirus from the culture supernatant. In embodiments, provided herein is a lentivirus produced by the aforementioned method.

[0014] In embodiments, provided herein is a method of expressing a GOI, comprising (i) inserting an expression cassette comprising a polynucleotide sequence encoding the GOI in the lentiviral transfer vector of the lentiviral vector system described herein; (ii) introducing the lentiviral vector system into a first cell; (iii) culturing the first cell under conditions where a lentivirus is produced; (iv) harvesting the lentivirus from the culture supernatant; (v) contacting a second cell with the lentivirus; and (vi) expressing the GOI. In embodiments, the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

[0015] In embodiments, provided herein is a method of expressing a GOI, comprising (i) introducing the lentiviral vector system described herein into a first cell; (ii) culturing the first cell under conditions where a lentivirus is produced; (iii) harvesting the lentivirus from the culture supernatant; (iv) contacting a second cell with the lentivirus; and (v) expressing the GOI. In embodiments, the second cell is in vitro. In embodiments, the second cell is in vivo.

[0016] In embodiments, provided herein is a kit comprising a lentiviral vector system; wherein the lentiviral vector system comprises: (a) a first packaging vector comprising a polynucleotide sequence encoding a Rev protein; (b) a second packaging vector comprising a polynucleotide sequence encoding a Gag protein and a Pol protein; (c) a third packaging vector comprising a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and (d) a transfer vector comprising, from 5’ to 3’: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; anda polyadenylation (pA) signal polynucleotide sequence. In embodiments, the transfer vector of the lentiviral vector system further comprises a splice donor site and a splice acceptor site. In embodiments, the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence. In embodiments, the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence. In embodiments, the A5’ LTR of the transfer vector of the lentiviral vector system comprises an R region and a U5 region of an HIV 5’ LTR. In embodiments, the A5’ LTR of the transfer vector of the lentiviral vector system consists of an R region and a U5 region of an HIV 5’ LTR. In embodiments, the HIV is HIV-1 subtype B. In embodiments, the 3’ LTR of the transfer vector of the lentiviral vector system is a self-inactivating 3’ LTR (SIN 3’ LTR). In embodiments, the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR. In embodiments, the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR. In embodiments, the HIV is HIV-1 subtype B. In embodiments, the A 5’ LTR and 3’ LTR of the transfervector ofthe lentiviral vector system are from the same HIV subtype. In embodiments, the pA of the transfer vector of the lentiviral vector system is a bovine growth hormone pA (bGH-pA). In embodiments, the location for inserting the expression cassette of the transfer vector ofthe lentiviral vector system is a multiple cloning site (MCS). In embodiments, the CMV promoter polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:1. In embodiments, the A5’ LTR polynucleotide sequence of the transfer vector ofthe lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:2. In embodiments, the 3’ LTR polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:3. In embodiments, the pA polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:4. In embodiments, the Psi (T) viral packaging signal polynucleotide sequence of the transfer vector ofthe lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:5. In embodiments, the AGag polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:6. In embodiments, the AEnv polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:7. In embodiments, the central polypurine tract (cPPT) polynucleotide sequence of the transfer vector ofthe lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:8. In embodiments, the polypurine tract (PPT) polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:9. In embodiments, the transfer vector of the lentiviral vector system comprises the polynucleotide sequence of SEQ ID NQ:10. In embodiments, the first packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:11. In embodiments, the second packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:12. In embodiments, the third packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:13.

[0017] In embodiments, provided herein is a kit comprising a lentiviral vector system, comprising: (i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13.

[0018] In embodiments, the lentiviral vector system of any of the kits provided herein is able to generate a crude titer of at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL. In embodiments, the lentiviral vector system of any of the kits provided herein is able to generate a crude titer of at least 1 E8 TU/mL. In embodiments, the lentiviral vector system of any of the kits provided herein is able to generate a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200. In embodiments, the lentiviral vector system of any of the kits provided herein is able to generate a P:l of at most 200. In embodiments, the transfer vector of the lentiviral vector system of any of the kits provided herein further comprises an expression cassette comprising a polynucleotide encoding the GOI. In embodiments, the expression cassette comprises an EF1a promoter polynucleotide sequence. In embodiments, the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide. In embodiments, the woodchuck hepatitis virus post-transcriptional regulatory element is an woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. In embodiments, the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39. In embodiments, the expression cassette comprises from 5’ to 3’: (i) an EF1a promoter polynucleotide sequence;(ii) a polynucleotide encoding the GOI; and (iii) a woodchuck hepatitis virus post-transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. In embodiments, the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

BRIEF DESCRIPTION OF THE FIGURES

[0019] Fig. 1 provides a schematic of the 3^rd generation lentiviral vector system described herein.

[0020] Fig. 2A - Fig. 2B provide exemplary schematics of the lentiviral vectors described herein. Fig. 2A provides exemplary schematics of the packaging vectors. Fig. 2B provides an exemplary schematic of the transfer vector. Ori = origin of replication; Kan-R = kanamycin selection cassette; RSV = RSV promoter; CMV = CMV promoter; HSV-Tk-pA = HSV thymidine kinase polyadenylation signal; A5’ LTR = truncated 5’ long terminal repeat; ip = Psi viral packaging signal sequence; cPPT = central polypurine tract; EF1a = EF1a promoter; GOI = gene of interest; oPRE = woodchuck hepatitis virus post- transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide; PPT = polypurine tract; SIN- AU3 = selfinactivating truncated 3’ LTR; bGH-pA = bovine growth hormone polyadenylation sequence.

[0021] Fig. 3 provides an exemplary schematic of a lentiviral transfer vector. The location of the AGag- AEnv-RRE sequence in the lentiviral transfer vector is indicated by a box. Various AGag-AEnv-RRE sequences were tested and optimized in Example 1 .

[0022] Fig. 4A - Fig. 4C show the functional outcomes observed with the modified AGag- AEnv-RRE sequences as described in Example 1 . Fig. 4A shows viral titers, Fig. 4B shows p24 levels, and Fig. 4C shows the P:l ratios.

[0023] Fig. 5A - Fig. 5C show schematic representations of packaging vectors evaluated in Example

2. Fig. 5A is a schematic representation of the pEB-00517 and pEB-00516 Rev-encoding packaging vectors. RSV = Promoter from Rous Sarcoma Virus; HIV Rev = Codon Optimized HIV-1 Rev gene, Ori= origin of replication; Kan-R = Kanamycin A gene; HIV-1-pA = poly A sequence from Human Immunodeficiency Virus-1 ; Rabbit p-globin pA= rabbit p-globin poly A sequence. Fig. 5B is a schematic representation of the pEB-00520 and pEB-00521 Gag-Pol-encoding packaging vectors. Ori= origin of replication; Kan-R = Kanamycin A gene; CMV= Cytomegalovirus Promoter; hup-globin intron= Intron from human beta-globin gene intron 1 ; HIV-Gag-Pol = polynucleotide sequence encoding a Gag protein and a Pol protein; RRE = Rev responsive element; Rabbit p-globin pA= rabbit p-globin poly A sequence; and hup-globin term/pA = terminator and poly A sequence from human beta-globin gene intron 1 . Fig. 5C is a schematic representation of the pEB-00518 and pEB-00519 VSV-G-encoding packaging vectors. Ori= origin of replication; Kan-R = Kanamycin A gene; CMV= Cytomegalovirus Promoter; human p-globin intron= Intron from human beta-globin gene intron 1 ; rabbit p-globin intron= Intron from rabbit beta-globin gene intron 1 ; VSV-G= vesicular stomatitis virus glycoprotein; and human p-globin pA = poly A sequence from human beta-globin gene intron 1 .

[0024] Figs. 6A - Fig. 6C show the functional outcomes observed with the different combinations of packaging vectors as outlined in Example 2. Fig. 6A shows viral titers, Fig. 6B shows p24 levels, and Fig. 6C shows the P:l ratios.

[0025] Fig. 7A - Fig. 7C show the functional outcomes observed with the different combinations of packaging vectors and lentiviral transfer vectors as outlined in Example 2. Fig. 7A shows viral titers, Fig. 7B shows p24 levels, and Fig. 7C shows the P:l ratios. “Commercial” refers to a commercial packaging vector set and a commercial lentiviral transfer vector.

[0026] Fig. 8A - Fig. 8C show the functional outcomes observed with Transfer Vector 1 (comprising a AGag-AEnv-RRE sequence of pEB-00500); Transfer Vector 2 (comprising a AGag-AEnv-RRE sequence of pEB-00509); or Transfer Vector 3 (comprising a AGag-AEnv-RRE sequence of pEB- 00511). The GOI for each transfer vector is a CD19 chimeric antigen receptor. The following lentiviral packaging vectors were used: the Gag-Pol Packaging Plasmid pEB-00521 ; a Rev Packaging Plasmid pEB-00516; and a VSV-G Packaging Vector comprising pEB-00519. Fig. 8A shows viral titers, Fig. 8B shows p24 levels, and Fig. 8C shows the P:l ratios.

[0027] Fig. 9A - Fig. 9C show the functional outcomes observed with Transfer Vector 1 (comprising a AGag-AEnv-RRE sequence of pEB-00500); Transfer Vector 2 (comprising a AGag-AEnv-RRE sequence of pEB-00509); or Transfer Vector 3 (comprising a AGag-AEnv-RRE sequence of pEB- 00511). The GOI for each transfer vector is a MAGEA1-TCR. The following lentiviral packaging vectors were used: the Gag-Pol Packaging Vector pEB-00521 ; a Rev Packaging Vector pEB-00516; and a VSV-G Packaging Vector comprising pEB-00519. Fig. 9A shows viral titers, Fig. 9B shows p24 levels, and Fig. 9C shows the P:l ratios.

[0028] Fig. 10A - Fig. 10C show the functional outcomes observed with Transfer Vector 1 (comprising a AGag-AEnv-RRE sequence of pEB-00500); Transfer Vector 2 (comprising a AGag-AEnv-RRE sequence of pEB-00509); or Transfer Vector 3 (comprising a AGag-AEnv-RRE sequence of pEB- 00511). The GOI for each transfer vector is a FLT3 CAR. The following lentiviral packaging vectors were used: the Gag-Pol Packaging Vector pEB-00521 ; a Rev Packaging Vector pEB-00516; and a VSV-G Packaging Vector comprising pEB-00519. Fig. 10A shows viral titers, Fig. 10B shows p24 levels, and Fig. 10C shows the P:l ratios.

[0029] Fig. 11 A - Fig. 11 C show the crude lentiviral titers observed with T ransfer Vector 1 (comprising a AGag-AEnv-RRE sequence of pEB-00500); Transfer Vector 2 (comprising a AGag-AEnv-RRE sequence of pEB-00509); or Transfer Vector 3 (comprising a AGag-AEnv-RRE sequence of pEB- 00511). The following lentiviral packaging vectors were used: the Gag-Pol Packaging Vector pEB- 00521 ; a Rev Packaging Vector pEB-00516; and a VSV-G Packaging Vector comprising pEB-00519. The GOI is CD19 CAR in Fig. 11 A. The GOI is MAGEA1-TCR in Fig. 11 B. The GOI is FLT3 CAR in Fig. 11C.

[0030] Fig. 12 shows the crude lentiviral titers observed when the polyA sequence in the Rev lentiviral packaging vector is varied, as outlined in Example 3.

[0031] Fig. 13A - Fig. 13C show the functional outcomes observed when the concentration of sodium butyrate and the ratio of lentiviral transfer vector: Gag/Pol Packaging Vector; Rev Packaging Vector; VSV-G Packaging Vector are varied. Fig. 13A shows viral titers, Fig. 13B shows p24 levels, and Fig. 13C shows the P:l ratios.

[0032] Fig. 14 shows modifications made to optimize the transcription start site of the CMV promoter in the lentiviral transfer vector design.

[0033] Fig. 15A - Fig. 15C shows the functional outcomes observed with the modified transcriptional start sites illustrated in Fig. 14. Fig. 15A shows viral titers, Fig. 15B shows p24 levels, and Fig. 15C shows the P:l ratios.

[0034] Fig. 16A - Fig. 16B provide comparisons of crude titers (Fig. 16A) and P:l ratios (Fig. 16B) achieved with an exemplary lentiviral vector system of the present disclosure (referred to in the figures as “pEB-00541 ”) and a commercially available lentiviral vector system (referred to in the figures as “Commercial”), using Flt3 CAR as an exemplary GOI.

[0035] Fig. 17A - Fig. 17B provide comparisons of crude titers (Fig. 17A) and P:l ratios (Fig. 17B) achieved with an exemplary lentiviral vector system of the present disclosure (referred to in the figures as “pEB-00541 ”) and a commercially available lentiviral vector system (referred to in the figures as “Commercial”), using GFP as an exemplary GOI.

DETAILED DESCRIPTION

Overview

[0036] There are several challenges in the manufacturing of commercial clinical grade lentiviral vectors including scaling of the production and purification process, achieving high viral titers, and producing high quality viral vectors (e.g., vectors with a low P:l ratio). Ideally, vector design and manufacturing processes will produce high viral titers (with a high number of viral particles coming from a single cell), a high percentage of infectious particles, and remain relatively free of other contaminants such as empty capsids or cellular debris. The lentiviral system described herein is designed to work in a scalable suspension cell process to produce increased titers over other commercial systems and result in a higher proportion of full capsids as measured by a low particle to infectious titer ratio. This solves the issues of scalability and provides a starting product with a higher yield, higher purity, and that is easier to purify.

[0037] Described herein is a novel, third generation, four vector lentiviral vector system comprising a transfer vector encoding a self-inactivating lentiviral vector genome and three packaging vectors encoding the Gag protein, the Pol protein, the Rev protein, and an Envelope protein (e.g., Vesicular Stomatitis Virus Glycoprotein (VSV-G)). This vector system combines novel or optimized sequences designed to increase overall production titer, to increase packaging of full length and functional lentiviral genomes, and to decrease empty capsid production (as measured by P:l ratios). The system described herein utilizes novel combinations of the Gag gene, Pol gene, Rev gene, Rev response element, and 5’ LTR that were screened in combination for their ability to increase overall virus titer and packaging. Further, the transfer vector in this system utilizes an optimized polynucleotide sequence at the junction of the transcriptional start site (TSS) and the truncated 5’ LTR that achieved precise transcription initiation at the R-U3 boundary (i.e., the resulting transcript contains triple Gs at the 5’ end) and surprisingly more functional lentiviral particles with higher infectivity. As a result, the system described herein generates increased viral titers, increased p24 level, and reduced P:l ratio, compared with a commercially available system.

[0038] The lentiviral system provided herein enables the production of clinical grade lentiviral vectors that have a more efficient packaging efficiency and are easier to purify due to a low particle to infectious titer ratio.

Definitions

[0039] The following terms are used in the description herein and the appended claims.

[0040] The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0041] The term “about” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide, dose, time, temperature, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1 %, ± 0.5%, or even ± 0.1 % of the specified amount.

[0042] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0043] The term “vector” refers to a carrier for a nucleic acid (i.e., a DNA or RNA molecule), which can be used to introduce the nucleic acid into a cell. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the nucleic acid it carries within the host cell.

[0044] An "expression cassette" is a polynucleotide sequence that comprises a sequence encoding a protein or an RNA and the necessary regulatory regions needed for expression of the sequence in a cell. In some embodiments, the sequence encoding a protein or an RNA is operably linked to another sequence in the cassette. [0045] The term "operably linked" means that the regulatory sequences necessary for expression of the sequence encoding a protein or an RNA are placed in the nucleic acid molecule in the appropriate positions relative to the sequence to effect expression of the protein or RNA.

[0046] The term “from 5’ to 3’,” when used in the context of polynucleotides, refers to a direction from the 5’ end of the polynucleotide to the 3’ end of the polynucleotide. In the context of double-stranded DNA, it refers to the direction based on the sense strand of the DNA.

[0047] The term “downstream” in the context of a polynucleotide sequence refers to a location in the 3’ direction relevant to a reference point in the polynucleotide. In the context of double-stranded DNA, the term “downstream” refers to the location based on the sense strand of the DNA. In the context of an RNA sequence, the term “downstream” refers to a portion of the RNA sequence that is located 3’ to a particular point on the RNA strand.

[0048] A second polynucleotide sequence located “immediately downstream” of a first polynucleotide sequence, as used herein, means that the 3’ end of the first polynucleotide sequence is covalently linked to the 5’ end of the second polynucleotide sequence through a phosphodiester bond, without any extra nucleotides between the 3’ end of the first polynucleotide sequence and the 5’ end of the second polynucleotide sequence. In the context of double-stranded DNA, this refers to the sense strand of the DNA.

[0049] The term “upstream” in the context of a polynucleotide sequence refers to location in the 5’ direction relevant to a reference point in the polynucleotide. In the context of double-stranded DNA, the term “upstream” refers to the location based on the sense strand of the DNA. In the context of an RNA sequence, the term “upstream” refers to a portion of the RNA sequence that is located 5’ to a particular point on the RNA strand.

[0050] The term “polynucleotide” as used herein refers to any polyribonucleotide, polydeoxyribonucleotide, or hybrid polyribo-polydeoxyribonucleotide, including naturally occurring polynucleotides, synthetic polynucleotides, or any chemically, enzymatically, or metabolically modified forms of naturally occurring polynucleotides. The term encompasses both single- and double-stranded molecules, including DNA-DNA, DNA-RNA, or RNA-RNA duplexes, as well as molecules that are a mixture of single- and double-stranded regions. Polynucleotides may contain any of the standard pyrimidine or purine bases (i.e., adenine, guanine, cytosine, thymine, uracil), as well as any modified or uncommon bases such as tritylated bases or inosine.

[0051] The term “transcription start site” or “TSS” refers to the location in a promoter where the first DNA nucleotide is transcribed into RNA. In some embodiments, the TSS is guanosine (G).

[0052] 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. A person of skill in the art would use known techniques to calculate the percent identity of a nucleic acid or protein described herein to an alternative nucleic acid or protein. For example, percentage identity can be calculated using the tool EMBOSS Needle, which is available online. The following default parameters may be used for EMBOSS Needle Pairwise alignment: Matrix = DNAfuil; Gap Open = 10; Gap Extension = 0.5; End Gap Penalty = false;

End Gap Open = 10; End Gap Extend = 0.5.

The term “AGag-AEnv-RRE” or “AGag-AEnv-RRE sequence” as used herein refers to the polynucleotide comprising a truncated Gag gene (AGag) and a truncated Env gene (AEnv), wherein the AEnv comprises a Rev Response Element (RRE).

Lentiviral transfer vectors

[0053] In some embodiments, the present disclosure provides a lentiviral transfer vector that is superior to known commercially available lentiviral transfer vectors. A transfer vector is a nucleic acid vector comprising an insertion point for an expression cassette comprising a polynucleotide encoding a gene of interest (GOI), which is packaged into lentiviral particles.

[0054] Applicants found that several elements of the lentiviral transfer vector affected lentiviral titers, p24 titers; and P:l ratio. First, Applicants discovered that the AGag-AEnv-RRE sequence of the transfer vector influenced lentiviral titers and P:l ratios. Applicants incorporated nine different AGag-AEnv-RRE sequences (SEQ ID NO: 14-22) into a lentiviral transfer vector (see Example 1) and discovered that selection of AGag-AEnv-RRE sequence influenced lentiviral titer and P:l ratio. Second, Applicants also unexpectedly found that the sequence at the junction of the transcriptional start site (TSS) and the A5’ LTR influenced lentiviral titer and P: I ratio. High crude titers were observed using a lentiviral vector comprising three 5’ guanosines (3G) at the 5’ terminus of the A5’ LTR and a CMV promoter lacking the TSS G. This is surprising in light of the study by Brown et al. described below.

[0055] Brown et al. investigated the influence of 5’ guanosines on the function of viral particles. Brown et al., Science 368, 413-417 (2020). Brown et al. found that HIV-1 transcript with 3 guanosines (3G) at the 5’ end mainly functions as mRNA for gene expression whereas transcript with one guanosine (1 G) at the 5’ end mainly functions as genomic RNA and is packaged into viral particles. Surprisingly, Applicants found that, using a transfer vector design described herein, transcripts with 3G at the 5’ end produced higher crude titer and more functional viral particles with higher infectivity, compared to transcript with 1 G at the 5’ end.

[0056] The lentiviral transfer vectors of the present disclosure comprise one or more of the following components: a) a cytomegalovirus (CMV) promoter lacking the transcription start site (TSS) G; b) immediately downstream of a) a lentivirus 5' long terminal repeat (LTR) polynucleotide sequence, wherein the first four nucleotides at the 5’ end of the LTR consist of GGGX, wherein X is A, T, or C; c) a location for the insertion of an expression cassette comprising a polynucleotide sequence encoding a gene of interest; d) a lentivirus 3' LTR; and e) a polyadenylation (pA) signal sequence. In some embodiments, the lentiviral transfer vectors of the present disclosure comprise each of a) - e). In some embodiments, the lentiviral transfer vector further comprises a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; and a polypurine tract (PPT) polynucleotide sequence. [0057] The lentiviral transfer vectors of the present disclosure comprise (a) a modified CMV promoter lacking the TSS G and (b) a lentivirus 5' LTR polynucleotide sequence, located immediately downstream of (a); wherein the first four nucleotides at the 5’ end of the LTR consist of GGGX, wherein X is A, T, or C. This unique design results in an increased viral titer (e.g., at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL) or a decreased P:l ratio (e.g., at most 500, 450, 400, 350, 300, 250, or 200) compared with other commercially available lentiviral vector systems. In some embodiments, this unique design results in increased viral titers (e.g., at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL) compared with other commercially available lentiviral vector systems. In certain embodiments, this unique design results in a decreased P:l ratio (e.g., at most 500, 450, 400, 350, 300, 250, or 200) compared with other commercially available lentiviral vector systems. In other embodiments, this unique design results in an increased viral titer (e.g., at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL) and a decreased P:l ratio (e.g., at most 500, 450, 400, 350, 300, 250, or 200) compared with other commercially available lentiviral vector systems.

[0058] In some embodiments, the CMV promoter lacking the TSS G comprises a nucleotide sequence as set forth in SEQ ID NO: 1 . In some embodiments, the CMV promoter lacking the TSS G consists of a nucleotide sequence as set forth in SEQ ID NO: 1. In embodiments, the CMV promoter lacking the TSS G comprises a nucleotide sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 1 . In embodiments, the CMV promoter lacking the TSS G comprises a sequence that is at least 90 % identical to the nucleic acid of SEQ ID NO: 1 . In embodiments, the CMV promoter lacking the TSS G comprises a sequence that is at least 95 % identical to the nucleic acid of SEQ ID NO: 1. In embodiments, the CMV promoter lacking the TSS G comprises a sequence that is at least 96 % identical to the nucleic acid of SEQ ID NO: 1 . In embodiments, the CMV promoter lacking the TSS G comprises a sequence that is at least 97 % identical to the nucleic acid of SEQ ID NO: 1 . In embodiments, the CMV promoter lacking the TSS G comprises a sequence that is at least 98 % identical to the nucleic acid of SEQ ID NO: 1. In embodiments, the CMV promoter lacking the TSS G comprises a sequence that is at least 99 % identical to the nucleic acid of SEQ ID NO: 1.

[0059] Any suitable lentiviral 5' LTR (and/or 3’LTR described below) can be utilized in the transfer vectors and packaging vectors described herein, including an LTR obtained from any lentivirus species, sub-species, strain or clade. This includes primate and non-primate lentiviruses. Specific examples of species, etc., include, but are not limited to, e.g., HIV-1 (including subspecies, clades, or strains, such as A, B, C, D, E, F, and G, R5 and R5X4 viruses, etc.), HIV-2 (including subspecies, clades, or strains, such as, R5 and R5X4 viruses, etc.), simian immunodeficiency virus (SIV), simian/human immunodeficiency virus (SHIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine-arthritis-encephalitis virus, Jembrana disease virus, ovine lentivirus, visna virus, and equine infectious anemia virus. Genomic sequence for such viruses are widely available, e.g., HIV-1 (NC_001802), HIV-2 (NC_001722), SIV (NC_001549), SIV-2 (NC_004455), Caprine arthritisencephalitis virus (NC_001463), Simian-Human immunodeficiency virus (NC_001870), FIV (NC_001482), Jembrana disease virus (NC_001654), ovine (NC_001511), Visna virus (NC_001452), Equine infectious anemia virus (NC_001450), and BIV (NC_0011413).

[0060] In some embodiments, the 5’ LTR is an HIV LTR. In some embodiments, the 5’ LTR is an HIV- 1 subtype B LTR. In some embodiments, the 5’ LTR is an HIV-1 subtype B LTR. In some embodiments, the 3’ LTR is an HIV-1 subtype B LTR. In some embodiments, the 5’ LTR and 3’ LTR are from the same HIV subtype.

[0061] The lentiviral 5' LTR comprises signals utilized in gene expression, including enhancer, promoter, transcription initiation (capping), transcription terminator, and polyadenylation and typically comprises U3, R, and U5 regions. The U3 region of the 5’ LTR contains enhancer, promoter and transcriptional regulatory signals, including RBEIII, NF-kB, Sp1 , AP-1 and/or GABP motifs. The TATA box is located about 25 base pairs from the beginning of the R sequence, depending on the species and strain from which the 5' LTR was obtained. A completely intact 5' LTR can be utilized, or a modified (e.gr truncated) copy can be utilized.

[0062] In some embodiments, the 5’ LTR is a truncated 5’ LTR (A5’ LTR). In some embodiments, the A5’ LTR comprises an R region and a U5 region of a 5’ LTR. In some embodiments, the A5’ LTR consists of an R region and a U5 region of a 5’ LTR. In some embodiments, the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR. In some embodiments, the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR. In some embodiments, the A5’ LTR comprises an R region and a U5 region of an HIV subtype B 5’ LTR. In some embodiments, the A5’ LTR consists of an R region and a U5 region of an HIV subtype B 5’ LTR. In some embodiments, the A5’ LTR sequence comprises SEQ ID NO: 2. In some embodiments, the A5’ LTR sequence consists of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 90 % identical to the nucleic acid of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 95 % identical to the nucleic acid of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 96 % identical to the nucleic acid of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 97 % identical to the nucleic acid of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 98 % identical to the nucleic acid of SEQ ID NO: 2. In embodiments, the A5’ LTR comprises a sequence that is at least 99 % identical to the nucleic acid of SEQ ID NO: 2.

[0063] The transfer vector can also include a lentiviral 3' LTR. The 3' LTR comprises U3, R, and U5 regions. The 3' LTR can be intact and native, but preferably it is modified. Preferably modifications include those that produce an LTR which retains a minimal amount of functional activity, e.g., transcriptional (promoter-enhancer) functional activity. Such transcriptional activity can be determined routinely, e.g., using a reporter gene. Examples of modifications that produce LTRs with reduced (as compared to the native 3' LTR) and minimal functional activity include, e.g., deletions which are 5' (upstream) to the TATA box in the U3 region. Such deletions can include, e.g., deletions or modifications of one or more of the following transcriptional regulatory sites, such as RBEIII, NF-kB, and/or Sp1 , as well as the PPT site. An example of a 3' LTR with minimal transcriptional activity includes a modified lentivirus 3' LTR that comprises TATA box sequence but lacks part of the U3 sequence located 5' to the said TATA box sequence or in which the sequences located 5’ to the TATA box are modified (deletion, substitution, addition) such they are not functionally active. For instance, NF-kB and Sp1 sites can be mutagenized to the point where they are inactive, and/or unable to bind to regulatory proteins. Deletions of the region upstream of the TATA box include from about 5, 10, 15, 20, 25, 30, 40, 50, etc., nucleotides from the T nucleotide of the TATA box. In some embodiments, TATA box is deleted. In other embodiments, PPT is retained. In yet other embodiments, TATA box is deleted, and PPT is retained. The amount of transcriptional activity that remains (when compared to the native LTR) can be, for example, from about 0.1-1 %, 0.1-2%, 0.1-5%, 0.1-10%, 0.1-20%, 0.1-25%, 0.5-5%, 0.5-10%, 0.5-20%, 0.5-25%; about 0.1 %; about 0.5%; about 1 %; about 2%; about 5%; about 7%, about 10%, etc.

[0064] The 5' end of the U3 region is necessary for integration (terminal dinucleotide + att sequence). Thus, the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted. In some embodiments, a portion (e.g., the first 34 nucleotides) of the 5’ of the U3 region is retained. In other embodiments, a portion (e.g., the first 34 nucleotides) of the 5’ of the U3 region corresponding to nucleotides 290 through 323 of the PV22 NEF gene is retained. In certain embodiments, a portion (e.g., the first 34 nucleotides) of the 5’ and a portion (e.g., the last 19 nucleotides) of the 3’ of the U3 region is retained. In other embodiments, a portion (e.g., the first 34 nucleotides) of the 5’ and a portion (e.g., the last 19 nucleotides) of the 3’ of the U3 region is retained, and the TATA box is deleted. A modified 3' LTR, however, can retain sequences outside the engineered U3 region, e.g., PPT, R, and U5. Further, the TAR element in the R region can be replaced with a heterologous TAR sequence from a different lentiviral species or subspecies.

[0065] Since viral transcription begins at the 3' end of the U3 region of the 5' LTR, these sequences are not part of the viral mRNA, and a copy thereof from the 3' LTR acts as template for the generation of both LTR's in the integrated provirus. If the 3' LTR copy of the U3 region is altered in a vector construct, the vector RNA still is produced from the intact 5' LTR in the producer cells but cannot propagate in target cells. Transduction of such a vector results in the inactivation of both LTR's in the progeny virus. Thus, the retrovirus is self-inactivating (SIN) and such vectors are known as SIN transfer vectors. See, e.g., Mitta et al., Nucl. Acid Res., 30(21):e113, 2002; Zufferey et al., J. Virol., 72:9873- 9880, 1998; U.S. Pat. No. 6,428,953 (Naldini et al.).

[0066] In some embodiments, the 3’ LTR is a self-inactivating (SIN) LTR. In some embodiments, the SIN 3’LTR comprises an R region and a U5 region of a 3’ LTR. In some embodiments, the SIN 3’LTR further comprises a portion of the U3 region of a 3’ LTR. In some embodiments, the SIN 3’LTR comprises an R region and a U5 region of an HIV 3’ LTR. In some embodiments, the SIN 3’LTR further comprises a portion ofthe U3 region of an HIV 3’ LTR. In some embodiments, the SIN 3’LTR comprises an R region and a U5 region of an HIV subtype B 3’ LTR. In some embodiments, the SIN 3’LTR further comprises a portion of the U3 region of an HIV subtype B 3’ LTR. In some embodiments, the portion of the U3 region comprises a portion of the 5’ and a portion of the 3’ of the U3 region. In other embodiments, the portion of the U3 region comprises the first 34 nucleotides at the 5’ and the last 19 nucleotides at the 3’ of the U3 region. In some embodiments, the portion of the U3 region is about 10, 20, 30, 40, 50 or more nucleic acids in length. In some embodiments, the SIN 3’LTR sequence comprises SEQ ID NO: 3. In some embodiments, the SIN 3’LTR sequence consists of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 90 % identical to the nucleic acid of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 95 % identical to the nucleic acid of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 96 % identical to the nucleic acid of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 97 % identical to the nucleic acid of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 98 % identical to the nucleic acid of SEQ ID NO: 3. In embodiments, the SIN 3’LTR comprises a sequence that is at least 99 % identical to the nucleic acid of SEQ ID NO: 3.

[0067] In some embodiments, the transfer vector comprises a packaging sequence (psi) downstream of the 5' LTR. The psi sequence is recognized by the NC domain of the Gag protein and is utilized to facilitate encapsulation of the polynucleotide encoding the GOI into the lentiviral particles. See, e.g., Lever et al., J. Virol. (1989), 63: 4085-4087; Amarasinghe et al., J. Mol. Bio. (2001), 314(5):961-970. The psi packaging sequence functions relatively autonomous of neighboring sequences and its position in the transfer vector can be determined by routine methods known in the art. See, e.g., Man and Baltimore, J. Virol., 54(2): 401-407, 1985, which use a reporter gene to optimize positioning of the packaging sequence. In some embodiments, the psi packaging sequence comprises SEQ ID NO: 5. In some embodiments, the psi packaging sequence consists of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 90 % identical to the nucleic acid of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 95 % identical to the nucleic acid of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 96 % identical to the nucleic acid of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 97 % identical to the nucleic acid of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 98 % identical to the nucleic acid of SEQ ID NO: 5. In embodiments, the psi packaging sequence is at least 99 % identical to the nucleic acid of SEQ ID NO: 5.

[0068] In addition, the transfer vector can comprise a Rev response element (RRE) sequence which can be located either upstream or downstream of a central poly-purine tract (cPPT) sequence. The cPPT is a sequence used for efficient nuclear import. Merten et al. Mol Ther Methods Clin Dev. 2016; 3: 16017. In certain embodiments, the RRE is located within a nucleic acid encoding a truncated Envelope protein (AEnv). The RRE or cPPT sequence can be derived from the native or non-native (heterologous) lentiviral vector sequences. In some embodiments, the cPPT sequence comprises SEQ ID NO: 8. In some embodiments, the cPPT sequence consists of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 90 % identical to the nucleic acid of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 95 % identical to the nucleic acid of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 96 % identical to the nucleic acid of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 97 % identical to the nucleic acid of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 98 % identical to the nucleic acid of SEQ ID NO: 8. In embodiments, the cPPT sequence is at least 99 % identical to the nucleic acid of SEQ ID NO: 8.

[0069] In some embodiments, the transfer vector comprises a nucleic acid encoding a truncated Gag protein (AGag). Vink et al. Mol Ther. 2017 Aug 2;25(8):1790-1804. The presence of the nucleic acid encoding AGag enables packaging into viral particles. In some embodiments, the nucleic acid encoding AGag comprises SEQ ID NO: 6. In some embodiments, the nucleic acid encoding AGag consists of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 90 % identical to the nucleic acid of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 95 % identical to the nucleic acid of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 96 % identical to the nucleic acid of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 97 % identical to the nucleic acid of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 98 % identical to the nucleic acid of SEQ ID NO: 6. In embodiments, the nucleic acid encoding AGag is at least 99 % identical to the nucleic acid of SEQ ID NO: 6.

[0070] In some embodiments, the transfer vector comprises a nucleic acid encoding a truncated Env protein (AEnv). Vink et al. Mol Ther. 2017 Aug 2;25(8):1790-1804. The presence of the nucleic acid encoding AEnv enables packaging into viral particles. In some embodiments, the nucleic acid encoding AEnv comprises SEQ ID NO: 7. In some embodiments, the nucleic acid encoding AEnv consists of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 90 % identical to the nucleic acid of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 95 % identical to the nucleic acid of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 96 % identical to the nucleic acid of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 97 % identical to the nucleic acid of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 98 % identical to the nucleic acid of SEQ ID NO: 7. In embodiments, the nucleic acid encoding AEnv is at least 99 % identical to the nucleic acid of SEQ ID NO: 7.

[0071] In embodiments, the transfer vector comprises a AGag-AEnv-RRE sequence. In embodiments, the AGag- AEnv-RRE sequence comprises a sequence selected from any one of SEQ ID NOS: 14-22 or a sequence that has at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % identity to any one of SEQ ID NOS: 14-22.

[0072] In some embodiments, the transfer vector comprises a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI. In some embodiments, the location is between the psi packaging sequence and the 3' LTR. In certain embodiments, the location is between the cPPT and the PPT. In some embodiments, the location is a multiple cloning site. The expression cassette is the sequence which is encapsulated into the complete lentiviral particles. Any heterologous sequence of interest can be inserted into the transfer vector without limitation, including, sequences coding for therapeutic proteins (e.g., chimeric antigen receptors, engineered TCRs, enzymes, antibodies, etc.), siRNA, anti-sense RNA, microRNAs, aptamers, ribozymes, any gene inhibitory or silencing sequence, one or more components of a CRISPR/Cas system, and any sequence which is to be delivered to a host cell via a lentiviral particle.

[0073] In some embodiments, the expression cassette comprises an internal promoter. The internal promoter can be any suitable promoter. Non-limiting examples of suitable promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focus forming virus (SFFV) promoter, long terminal repeats (LTRs) from retrovirus (e.g., a Moloney murine leukemia virus (MoMLV) LTR promoter or a Rous sarcoma virus (RSV) LTR), a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH) promoter, a ferritin L (FerL) promoter, a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, a heat shock 70kDa protein 5 (HSPA5) promoter, a heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, a heat shock protein 70kDa (HSP70) promoter, a p-kinesin (p-KIN) promoter, the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken p-actin (CAG) promoter, a p-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter, and mouse metallothionein-l. Any of the promoters mentioned above can be utilized to drive expression of the GOI. In some embodiments, the internal promoter is an EF1 a promoter.

[0074] In some embodiments, the expression cassette comprises (i) an EF1 a promoter polynucleotide sequence; (ii) a polynucleotide encoding the GOI; and (iii) an woodchuck hepatitis virus post- transcriptional regulatory element variant 1 polynucleotide or woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide. [0075] In addition, the transfer vector can further comprise transcription termination signals, such as a polyA (pA) signal that is effective to terminate transcription driven by the promoter sequence. Any suitable polyA sequence can be utilized, e.g., sequences from beta globin (mammalian, human, rabbit, etc), thymidine kinase, growth hormone, SV40, and many others. In some embodiments, the pA is a bovine growth hormone pA. In some embodiments, the bovine growth hormone pA polynucleotide sequence comprises SEQ ID NO: 4. In some embodiments, the bovine growth hormone pA polynucleotide sequence consists of SEQ ID NO: 4.

[0076] In some embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter polynucleotide sequence lacking the transcription start site (TSS) G; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; (c) a Psi (T) viral packaging signal polynucleotide sequence; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag); (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv) wherein the AEnv comprises a Rev response element (RRE); (f) a central polypurine tract (cPPT) polynucleotide sequence; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence; (i) a 3’ LTR polynucleotide sequence; and (j) a polyadenylation (pA) signal polynucleotide sequence. In some embodiments, the transfer vector further comprises an origin of replication site (ORI). The origin of replication can be used to increase the copy number of the vector when present in a host cell. The SV40 ori is commonly used for this purpose. In other embodiments, the transfer vector further comprises a selection marker. Any selection markers known by a person with ordinary skills in the art can be used.

[0077] In some embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter lacking the TSS G, comprising a polynucleotide sequence at least 90% identical to SEQ ID NO: 1 ; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, at least 90% identical to SEQ ID NO: 2; (c) a Psi (T) viral packaging signal polynucleotide sequence at least 90% identical to SEQ ID NO: 5; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag), wherein the polynucleotide sequence is at least 90% identical to SEQ ID NO: 6; (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE), wherein the polynucleotide sequence is at least 90% identical to SEQ ID NO: 7; (f) a central polypurine tract (cPPT) polynucleotide sequence at least 90% identical to SEQ ID NO: 8; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence at least 90% identical to SEQ ID NO: 9; (i) a 3’ LTR polynucleotide sequence at least 90% identical to SEQ ID NO: 3; and (j) a bovine growth hormone polyadenylation (pA) signal polynucleotide sequence at least 90% identical to SEQ ID NO: 4.

[0078] In some embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter lacking the TSS G, comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 1 ; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, at least 95% identical to SEQ ID NO: 2; (c) a Psi (T) viral packaging signal polynucleotide sequence at least 95% identical to SEQ ID NO: 5; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag), wherein the polynucleotide sequence is at least 95% identical to SEQ ID NO: 6; (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE), wherein the polynucleotide sequence is at least 95% identical to SEQ ID NO: 7; (f) a central polypurine tract (cPPT) polynucleotide sequence at least 95% identical to SEQ ID NO: 8; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence at least 95% identical to SEQ ID NO: 9; (i) a 3’ LTR polynucleotide sequence at least 95% identical to SEQ ID NO: 3; and (j) a bovine growth hormone polyadenylation (pA) signal polynucleotide sequence at least 95% identical to SEQ ID NO: 4.

[0079] In some embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter lacking the TSS G, comprising a polynucleotide sequence at least 98% identical to SEQ ID NO: 1 ; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, at least 98% identical to SEQ ID NO: 2; (c) a Psi (T) viral packaging signal polynucleotide sequence at least 98% identical to SEQ ID NO: 5; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag), wherein the polynucleotide sequence is at least 98% identical to SEQ ID NO: 6; (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE), wherein the polynucleotide sequence is at least 98% identical to SEQ ID NO: 7; (f) a central polypurine tract (cPPT) polynucleotide sequence at least 98% identical to SEQ ID NO: 8; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence at least 98% identical to SEQ ID NO: 9; (i) a 3’ LTR polynucleotide sequence at least 98% identical to SEQ ID NO: 3; and (j) a bovine growth hormone polyadenylation (pA) signal polynucleotide sequence at least 98% identical to SEQ ID NO: 4.

[0080] In some embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter lacking the TSS G, comprising a polynucleotide sequence at least 99% identical to SEQ ID NO: 1 ; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, at least 99% identical to SEQ ID NO: 2; (c) a Psi (T) viral packaging signal polynucleotide sequence at least 99% identical to SEQ ID NO: 5; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag), wherein the polynucleotide sequence is at least 99% identical to SEQ ID NO: 6; (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE), wherein the polynucleotide sequence is at least 99% identical to SEQ ID NO: 7; (f) a central polypurine tract (cPPT) polynucleotide sequence at least 99% identical to SEQ ID NO: 8; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence at least 99% identical to SEQ ID NO: 9; (i) a 3’ LTR polynucleotide sequence at least 99% identical to SEQ ID NO: 3; and (j) a bovine growth hormone polyadenylation (pA) signal polynucleotide sequence at least 99% identical to SEQ ID NO: 4.

[0081] In some embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter lacking the TSS G, comprising a polynucleotide sequence as set forth in SEQ ID NO: 1 ; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, comprising SEQ ID NO: 2; (c) a Psi (T) viral packaging signal polynucleotide sequence comprising SEQ ID NO: 5; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag), wherein the polynucleotide sequence comprises SEQ ID NO: 6; (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv) wherein the AEnv comprises a Rev response element (RRE), wherein the polynucleotide sequence comprises SEQ ID NO: 7; (f) a central polypurine tract (cPPT) polynucleotide sequence comprising SEQ ID NO: 8; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence comprising SEQ ID NO: 9; (i) a 3’ LTR polynucleotide sequence comprising SEQ ID NO: 3; and (j) a bovine growth hormone polyadenylation (pA) signal polynucleotide sequence comprising SEQ ID NO: 4.

[0082] In other embodiments, the transfer vector comprises, from 5’ to 3’, (a) a CMV promoter lacking the TSS G, consisting of a polynucleotide sequence as set forth in SEQ ID NO: 1 ; (b) a A5’ LTR polynucleotide sequence immediately downstream of the CMV promoter, consisting of SEQ ID NO: 2; (c) a Psi (T) viral packaging signal polynucleotide sequence consisting of SEQ ID NO: 5; (d) a polynucleotide sequence encoding a truncated Gag protein (AGag), wherein the polynucleotide sequence consists of SEQ ID NO: 6; (e) a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE), wherein the polynucleotide sequence consists of SEQ ID NO: 7; (f) a central polypurine tract (cPPT) polynucleotide sequence consisting of SEQ ID NO: 8; (g) a location for inserting an expression cassette comprising a polynucleotide sequence encoding a GOI; (h) a polypurine tract (PPT) polynucleotide sequence consisting of SEQ ID NO: 9; (i) a 3’ LTR polynucleotide sequence consisting SEQ ID NO: 3; and (j) a bovine growth hormone polyadenylation (pA) signal polynucleotide sequence consisting of SEQ ID NO: 4.

[0083] In some embodiments, the lentiviral transfer vector comprises SEQ ID NO: 10. In some embodiments, the lentiviral transfer vector consists of SEQ ID NO: 10. In other embodiments, the lentiviral transfer vector comprises a polynucleotide sequence that is at least 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence set forth in SEQ ID NO: 10. A schematic of an exemplary transfer vector is provided in Fig. 2B and Fig. 3.

[0084] One or more splice donor (SD) sites can be present in the transfer vector. A splice donor site is typically present between the 3' end of the 5' LTR and the packaging sequence. A downstream splice acceptor (SA) can also be present. The SD site can be present in multiple copies at any effective locations in the vector. The SD can have a native lentiviral sequence, or it can be a mutated copy of it. In some embodiments, the lentiviral transfer vector further comprises a splice acceptor and a splice donor site. In some embodiments, the splice donor site is located within the psi packaging polynucleotide sequence. In some embodiments, the splice acceptor site is located between the AEnv sequence and the cPPT sequence.

Lentiviral packaging vectors

[0085] In another aspect, the present disclosure provides lentiviral packaging vectors that are superior to commercially available lentiviral packaging vectors. These packaging vectors contain the additional elements for producing a functional lentiviral particle in a compatible host cell, include structural proteins (e.g., the Gag protein), processing proteins (e.g., the Pol protein), regulator proteins (e.g., the Rev protein), envelope proteins (e.g., the VSV-G protein), and the expression and regulatory signals needed to manufacture the proteins in host cells and assemble functional viral particles. Schematics of exemplary packaging vectors are provided in Fig. 2A and Figs. 5A-5C.

[0086] In some embodiments, the present disclosure provides a lentiviral packing vector comprising a polynucleotide sequence encoding the Rev protein (referred to herein as “Rev Packaging Vector”). In some such embodiments, the lentiviral packing vector may comprise, from 5’ to 3’, a promoter polynucleotide sequence operably linked to the polynucleotide encoding the Rev protein and a polyadenylation polynucleotide sequence. In some such embodiments, the lentiviral packing vector may comprise, from 5’ to 3’, an RSV promoter polynucleotide sequence operably linked to the polynucleotide encoding the Rev protein and an HSV thymidine kinase polyadenylation polynucleotide sequence. In some embodiments, the Rev Packaging Vector comprises SEQ ID NO: 11. In other embodiments, the Rev Packaging Vector consists of SEQ ID NO: 11 . In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 11 . In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 90 % identical to the nucleic acid of SEQ ID NO: 11. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 95 % identical to the nucleic acid of SEQ ID NO: 1 1 . In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 96 % identical to the nucleic acid of SEQ ID NO: 11 . In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 97 % identical to the nucleic acid of SEQ ID NO: 11 . In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 98 % identical to the nucleic acid of SEQ ID NO: 11. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 99 % identical to the nucleic acid of SEQ ID NO: 11 .

[0087] In some embodiments, the Rev Packaging Vector comprises SEQ ID NO: 28. In other embodiments, the Rev Packaging Vector consists of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 90 % identical to the nucleic acid of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 95 % identical to the nucleic acid of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 96 % identical to the nucleic acid of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 97 % identical to the nucleic acid of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 98 % identical to the nucleic acid of SEQ ID NO: 28. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 99 % identical to the nucleic acid of SEQ ID NO: 28.

[0088] In some embodiments, the Rev Packaging Vector comprises SEQ ID NO: 25. In other embodiments, the Rev Packaging Vector consists of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 90 % identical to the nucleic acid of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 95 % identical to the nucleic acid of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 96 % identical to the nucleic acid of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 97 % identical to the nucleic acid of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 98 % identical to the nucleic acid of SEQ ID NO: 25. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 99 % identical to the nucleic acid of SEQ ID NO: 25.

[0089] In some embodiments, the Rev Packaging Vector comprises SEQ ID NO: 33. In other embodiments, the Rev Packaging Vector consists of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 90 % identical to the nucleic acid of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 95 % identical to the nucleic acid of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 96 % identical to the nucleic acid of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 97 % identical to the nucleic acid of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 98 % identical to the nucleic acid of SEQ ID NO: 33. In embodiments, the nucleic acid sequence of the Rev Packaging Vector is at least 99 % identical to the nucleic acid of SEQ ID NO: 33.

[0090] A schematic of exemplary lentiviral packing vectors comprising a polynucleotide sequence encoding the Rev protein is provided in Fig. 2A and Fig. 5A.

[0091] In some embodiments, the present disclosure provides a lentiviral packing vector comprising a polynucleotide sequence encoding a Gag and a Pol protein (also referred to herein as “Gag-Pol Packaging Vector”). The gag and pol coding sequences can be organized as the Gag-Pol Precursor in native lentivirus. The gag sequence codes for a 55-kD Gag precursor protein, also called p55. The p55 is cleaved by the virally encoded protease4 (a product of the pol gene) during the process of maturation into four smaller proteins designated MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved away from Gag by a virally encoded protease, and further digested to separate the protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities. Native Gag-Pol sequences can be utilized in the packaging vector, or modifications can be made, such as chimeric Gag-Pol, where the Gag and Pol sequences are obtained from different viruses (e.g., different species, subspecies, strains, clades, etc.), and/or where the sequences have been modified to improve transcription and/or translation, and/or reduce recombination.

[0092] The packaging vector can further comprise an RRE element, including an RRE element which is obtained from a different lentiviral species than the gag and pol sequences. The RRE element is the binding site forthe Rev polypeptide which is a 13-kD sequence-specific RNA binding protein. Constructs which contain the RRE sequence depend on the Rev polypeptide for efficient expression. Rev binds to a 240-base region of complex RNA secondary structure of the RRE that is located within the second intron of HIV, distal to the pol and gag coding sequences. The binding of Rev to RRE facilitates the export of unspliced and incompletely spliced viral RNAs from the nucleus to the cytoplasm, thereby regulating the expression of HIV proteins. The RRE element can be in any suitable position on the construct, preferably following the Gag-Pol precursor in its approximate native position.

[0093] In some embodiments, the lentiviral packing vector may comprise, from 5’ to 3’, a promoter polynucleotide sequence, an intron sequence, a polynucleotide sequence encoding the Gag and Pol proteins and an RRE, and a polyadenylation polynucleotide sequence. In some such embodiments, the lentiviral packing vector may comprise, from 5’ to 3’, a CMV promoter polynucleotide sequence, a human p-globin intron sequence, a polynucleotide sequence encoding the Gag and Pol proteins and an RRE, and a rabbit p-globin polyadenylation polynucleotide sequence. In some embodiments, the sequence of the Gag-Pol Packaging Vector comprises SEQ ID NO: 12. In some embodiments, the sequence of the Gag-Pol Packaging Vector consists of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 90 % identical to the nucleic acid of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 95 % identical to the nucleic acid of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 96 % identical to the nucleic acid of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 97 % identical to the nucleic acid of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 98 % identical to the nucleic acid of SEQ ID NO: 12. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 99 % identical to the nucleic acid of SEQ ID NO: 12.

[0094] In some embodiments, the sequence of the Gag-Pol Packaging Vector comprises SEQ ID NO: 26. In some embodiments, the sequence of the Gag-Pol Packaging Vector consists of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 90 % identical to the nucleic acid of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag- Pol Packaging Vector is at least 95 % identical to the nucleic acid of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 96 % identical to the nucleic acid of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 97 % identical to the nucleic acid of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 98 % identical to the nucleic acid of SEQ ID NO: 26. In embodiments, the nucleic acid sequence of the Gag-Pol Packaging Vector is at least 99 % identical to the nucleic acid of SEQ ID NO: 26.

[0095] A schematic of an exemplary lentiviral packing vector comprising a polynucleotide sequence encoding the Gag and Pol proteins is provided in Fig. 2A and Fig 5B.

[0096] In some embodiments, the present disclosure provides a lentiviral packing vector comprising a polynucleotide sequence encoding an envelope protein. The envelope polypeptide is displayed on the viral surface and is involved in the recognition and infection of host cells by a virus particle. The host range and specificity can be changed by modifying or substituting the envelope polypeptide, e.g., with an envelope expressed by a different (heterologous) viral species or which has otherwise been modified, referred to as pseudotyping. See, e.g., Yee et al., Proc. Natl. Acad. Sci. USA 91 : 9564-9568, 1994. Vesicular stomatitis virus (VSV) protein G (VSV-G) has been used extensively because of its broad species and tissue tropism and its ability to confer physical stability and high infectivity to vector particles. See, e.g., Yee et al, Methods Cell Biol., (1994) 43:99-112. Lentiviral packaging vectors comprising a VSV-G envelope protein are referred to herein as “VSV-G Packaging Plasmid.”

[0097] The envelope protein may be an envelope protein of a human immunodeficiency virus-1 (HIV- 1), Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), Rous sarcoma virus (RSV), respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), hepatitis virus, influenza virus, moloka virus, Rabies virus, filovirus, Ebola virus, Marburg virus, an amphotropic virus, or an alphavirus. In embodiments, the envelope protein is hemagglutinin (HA), neuraminidase (NA), matrix protein 2 (M2), NB protein, BM2 protein, glycoprotein 1 , glycoprotein 2.

[0098] In aspects, the envelope protein is an envelope protein from Togaviridae, Rhabdoviridae, Retroviridae, Poxyiridae, Paramyxoviridae, or another enveloped virus family. Furthermore, a viral envelope protein can be modified or engineered to contain polypeptide sequences that allow the transduction vector to target and infect host cells outside its normal range or more specifically limit transduction to a cell or tissue type. For example, the envelope protein can be joined in-frame with targeting sequences, such as receptor ligands, antibodies (using an antigen-binding portion of an antibody or a recombinant antibody-type molecule, such as a single chain antibody), and polypeptide moieties or modifications thereof (e.g., where a glycosylation site is present in the targeting sequence) that, when displayed on the transduction vector coat, facilitate directed delivery of the virion particle to a target cell of interest. Furthermore, envelope proteins can further comprise sequences that modulate cell function. Modulating cell function with a transducing vector may increase or decrease transduction efficiency for certain cell types in a mixed population of cells.

[0099] In some embodiments, the lentiviral packing vector may comprise, from 5’ to 3’, a promoter polynucleotide sequence, an intron sequence, a polynucleotide sequence encoding the envelope protein, and a polyadenylation polynucleotide sequence. In some such embodiments, the lentiviral packing vector may comprise, from 5’ to 3’, a CMV promoter polynucleotide sequence, a rabbit p-globin intron sequence, a polynucleotide sequence encoding the VSV-G envelope protein, and a human p- globin polyadenylation polynucleotide sequence. In some embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein comprises SEQ ID NO: 13. In some embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein consists of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 90 % identical to the nucleic acid of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 95 % identical to the nucleic acid of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 96 % identical to the nucleic acid of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 97 % identical to the nucleic acid of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 98 % identical to the nucleic acid of SEQ ID NO: 13. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 99 % identical to the nucleic acid of SEQ ID NO: 13.

[0100] In some embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein comprises SEQ ID NO: 29. In some embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein consists of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % identical to the nucleic acid of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 90 % identical to the nucleic acid of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 95 % identical to the nucleic acid of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 96 % identical to the nucleic acid of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 97 % identical to the nucleic acid of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 98 % identical to the nucleic acid of SEQ ID NO: 29. In embodiments, the lentiviral packing vector comprising a polynucleotide sequence encoding the envelope protein is at least 99 % identical to the nucleic acid of SEQ ID NO: 29.

[0101] A schematic of an exemplary lentiviral packing vector comprising a polynucleotide sequence encoding the VSV-G protein is provided in Fig. 2A and Fig. 5C.

[0102] Any of the sequences which are present in the constructs of the present invention can be modified from their native form, e.g., to improve transcription, to improve translation, to reduce or alter secondary RNA structure, and/or to decrease recombination. Modifications include, e.g., nucleotide addition, deletion, substitution, and replacements. For example, coding sequences for Gag-Pol, Rev, and VSV-G packaging vectors can be modified by replacing naturally-occurring codons with non- naturally-occurring codons, e.g., to improve translation in a host cell by substituting them with codons which are translated more effectively in the host cell. The host cell can be referred to as a compatible cell, e.g., to indicate the sequence modification has its effect when the sequence is expressed in a particular host cell type. In addition, sequences can be modified to remove regulatory elements, such as the packaging sequence. Sequences can also be altered to eliminate recombination sites. Examples of hot spots for recombination are, e.g., disclosed in Zhuang et al., J. Virol., 76:1 1273-1 1282, 2002.

Lentiviral Vector Systems

[0103] In yet another aspect, the present disclosure provides a lentiviral vector system that is superior to commercially available lentiviral vector systems for producing fully-enveloped and functional lentiviral particles. In some embodiments, the lentiviral vector system comprises a lentiviral transfer vector and one or more packaging vectors described herein. In some embodiments, the lentiviral vector system comprises a lentiviral transfer vector and two packaging vectors described herein. In some embodiments, the lentiviral vector system comprises a lentiviral transfer vector and three packaging vectors described herein. In some embodiments, the lentiviral vector system comprises a lentiviral transfer vector and four packaging vectors described herein. A schematic of the systems described herein is provided in Fig. 1.

[0104] In some embodiments, the present disclosure provides a lentiviral vector system comprising: (a) a first packaging vector comprising a polynucleotide sequence encoding a Rev protein; (b) a second packaging vector comprising a polynucleotide sequence encoding a Gag protein and a Pol protein; (c) a third packaging vector comprising a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and (d) a transfer vector comprising a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G and a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C. The packaging and transfer vectors can comprise any of the modifications or elements described above. [0105] In some embodiments, one of the first, the second, and the third packaging vectors is a packaging vector described herein, and the rest of the packaging vectors is a packaging vectors that a person in the art would reasonably use. In some embodiments, two of the first, the second, and the third packaging vectors are packaging vectors described herein, and the rest of the packaging vector is any packaging vectors that a person in the art would reasonably use. In some embodiments, all of the first, the second, and the third packaging vectors are packaging vectors described herein, and the transfer vector is any transfer vector that a person in the art would reasonably use.

[0106] In some embodiments, the present disclosure provides a lentiviral vector system comprising:

(a) a first packaging vector comprising from 5’ to 3’, an RSV promoter polynucleotide sequence operably linked to a polynucleotide encoding a Rev protein and an HSV thymidine kinase polyadenylation polynucleotide sequence;

(b) a second packaging vector comprising from 5’ to 3’, a CMV promoter polynucleotide sequence, a human p-globin intron sequence, a polynucleotide sequence encoding a Gag protein and a Pol protein and an RRE, and a rabbit p-globin polyadenylation polynucleotide sequence;

(c) a third packaging vector comprising, from 5’ to 3’, a CMV promoter polynucleotide sequence, a rabbit p-globin intron sequence, a polynucleotide sequence encoding a VSV-G protein, and a human p-globin polyadenylation polynucleotide sequence; and

(d) a transfer vector.

[0107] In some embodiments, the present disclosure provides a lentiviral vector system comprising:

(d) a transfer vector comprising a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C.

[0108] In some embodiments, the present disclosure provides a lentiviral vector system comprising:

(a) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ;

(b) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12;

(c) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13; and

(d) a transfer vector. [0109] In some embodiments, the present disclosure provides a lentiviral vector system comprising:

(d) a transfer vector comprising from 5’ to 3’, a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C.

[0110] In some embodiments, the present disclosure provides a lentiviral vector system comprising:

(d) a transfer vector comprising from 5’ to 3’, a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

[0111] In some embodiments, the present disclosure provides a lentiviral vector system comprising:

(a) a first packaging vector comprising from 5’ to 3’, an RSV promoter polynucleotide sequence operably linked to a polynucleotide encoding a Rev protein and an HSV thymidine kinase polyadenylation polynucleotide sequence.;

(d) a transfer vector comprising from 5’ to 3’, a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi ( ¹) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

[0112] In some embodiments, the lentiviral vector system of the present disclosure comprises: (i) a transfer vector comprising a polynucleotide sequence of SEQ ID NO:10; (ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13.

[0113] In certain embodiments, the lentiviral vector system of the present disclosure comprises: (i) a transfer vector consisting of a polynucleotide sequence of SEQ ID NO: 10; (ii) a first packaging vector consisting of a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector consisting of a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector consisting of a polynucleotide sequence of SEQ ID NO:13.

[0114] In other embodiments, the lentiviral vector system of the present disclosure consists of: (i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13.

[0115] In yet other embodiments, the lentiviral vector system of the present disclosure consists of: (i) a transfer vector consisting of a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector consisting of a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector consisting of a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector consisting of a polynucleotide sequence of SEQ ID NO:13.

Kits

[0116] In still another aspect, the present disclosure provides a kit comprising a lentiviral vector system described herein. In some embodiments, the kit comprises a lentiviral transfer vector and one or more packaging vectors described herein. In some embodiments, the kit comprises a lentiviral transfer vector and two packaging vectors. In some embodiments, the kit comprises a lentiviral transfer vector and three packaging vectors. In some embodiments, the kit comprises a lentiviral transfer vector and four packaging vectors.

[0117] In some embodiments, the kit comprises one, two, or three packaging vectors described herein and a lentiviral transfer vector described herein. In some embodiments, the kit comprises one, two, or three packaging vectors described herein and one, two, or three packaging vectors that a person in the art would reasonably use. In some embodiments, the kit comprises one packaging vector described herein and the rest of the packaging vectors are those that a person in the art would reasonably use. In some embodiments, the kit comprises two packaging vectors described herein and the rest of the packaging vectors are those that a person in the art would reasonably use. In some embodiments, the kit comprises a first, second, and third packaging vector described herein and a transfer vector, which is any transfer vector that a person in the art would reasonably use.

[0118] In some embodiments, the present disclosure provides a kit comprising: (a) a first packaging vector comprising a polynucleotide sequence encoding a Rev protein; (b) a second packaging vector comprising a polynucleotide sequence encoding a Gag protein and a Pol protein; (c) a third packaging vector comprising a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and (d) a transfer vector comprising a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G and a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four- nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C. The packaging and transfer vectors can comprise any of the modifications or elements described above.

[0119] In some embodiments, the present disclosure provides a kit comprising:

[0120] In some embodiments, the present disclosure provides a kit comprising a lentiviral vector system comprising: (a) a first packaging vector comprising from 5’ to 3’, an RSV promoter polynucleotide sequence operably linked to a polynucleotide encoding a Rev protein and an HSV thymidine kinase polyadenylation polynucleotide sequence;

(d) a transfer vector.

[0121] In some embodiments, the present disclosure provides a kit comprising a lentiviral vector system comprising:

[0122] In some embodiments, the present disclosure provides a kit comprising a lentiviral vector system comprising:

(d) a transfer vector.

[0123] In some embodiments, the present disclosure provides a kit comprising a lentiviral vector system comprising:

(c) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13; and (d) a transfer vector comprising from 5’ to 3’, a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C.

[0124] In some embodiments, the present disclosure provides a kit comprising a lentiviral vector system comprising:

[0125] In some embodiments, the kit of the present disclosure comprises: (i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13.

[0126] In certain embodiments, the kit of the present disclosure comprises: (i) a transfer vector consisting of a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector consisting of a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector consisting of a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector consisting of a polynucleotide sequence of SEQ ID NO:13.

[0127] In other embodiments, the kit of the present disclosure consists of: (i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ; (iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13.

[0128] In yet other embodiments, the kit of the present disclosure consists of: (i) a transfer vector consisting of a polynucleotide sequence of SEQ ID NQ:10; (ii) a first packaging vector consisting of a polynucleotide sequence of SEQ ID NO:11 ; (Hi) a second packaging vector consisting of a polynucleotide sequence of SEQ ID NO:12; and (iv) a third packaging vector consisting of a polynucleotide sequence of SEQ ID NO:13.

[0129] In some embodiments, the kit comprises one or more components of lentiviral vector system described herein. In some embodiments, a kit comprising one or more components of a lentiviral vector system described herein and further comprises one or more additional reagents, where such additional reagents can be selected from: a buffer; a control reagent; a control vector; a control lentiviral vector system and the like. Components of a kit can be in separate containers or can be combined in a single container.ln addition to above-mentioned components, in some embodiments a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (/.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

Compositions

[0130] In still another aspect, the present disclosure provides a composition of lentiviral particles produced by the methods described herein. The term “composition” as used herein refers to a formulation of lentiviral particles produced by the methods described herein that is capable of being administered or delivered to a subject or cell. Typically, formulations include all physiologically acceptable compositions with any pharmaceutically acceptable carriers, diluents, and/or excipients.

[0131] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Except insofar as any conventional media and/or agent is incompatible with the agents of the present disclosure, its use in compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[0132] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0133] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0134] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

Manufacture of Viral Particles and Methods of Use

[0135] In yet still another aspect, the present disclosure provides lentiviral particles, methods of producing lentiviral particles utilizing the lentiviral vector systems described herein. In other embodiments, the present disclosure provides methods of expressing a GOI using the lentiviral vector systems described herein.

[0136] In general, the lentiviral vector systems are introduced into host cells, which then produce the functional lentiviral particles. Examples of host cells which can be utilized to produce the lentiviral particles, include, any mammalian or human cell line or primary cell. Non-limiting examples include, e.g., HT1080, Jurkat, SupT1 , Hela, VERO, L929, BHK, NIH 3T3, MRC-5, BAE-1 , HEP-G2, NSO, U937, Namalwa, HL60, WEHI 231 , YAC 1 , U 266B1 , SH-SY5Y, CHO, e.g., CHO-K1 (CCL-61), 293.

[0137] The present invention provides methods for producing lentiviral particles comprising, e.g.,

(i) introducing the lentiviral vector system described herein into a cell; (ii) culturing the cell under conditions where a lentivirus is produced; and (iii) harvesting the lentivirus from the culture supernatant.

[0138] Any suitable methods for introducing the lentiviral vector system into a cell can be used in the particle manufacturing process including electroporation, calcium phosphate transfection, PEI polymer mediated transfection, fecturin, or lipid-based transfection methods. The lentiviral particles are then secreted into the cell culture medium where they can be recovered and optionally enriched or purified.

[0139] In some embodiments, the cell line utilized to manufacture the lentiviral particles can be modified to enhance vector protein production, e.g., by the introduction of RNAi or antisense to knockout genes that reduce the expression of genes that limit vector production, or by the introduction of sequences that enhance vector production. Sequences that code for cellular or viral enhancers can also be engineered into cell lines (e.g., using additional vectors), such as herpes virus, hepatitis B virus, which act on HIV LTRs to enhance the level of virus product, or cellular transactivator proteins. Cellular transactivation proteins include, e.g., NF-kB, UV light responsive factors, and T cell activation factors.

[0140] Producer cells are cultured under conditions effective to produce lentiviral particles including e.g., appropriate buffers, oxidizing agents, reducing agents, pH, co-factors, temperature, ion concentrations, suitable age and/or stage of cell (such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed) where cells are being used, culture conditions (including cell media, substrates, oxygen, carbon dioxide, glucose and other sugar substrates, serum, growth factors, etc.). Each of these parameters can be adjusted for optimal lentiviral particle production.

[0141] In some embodiments, the lentiviral particles produced according to the description herein are concentrated and/or purified, such as by flow-through ultracentrifugation, high-speed centrifugation, and tangential flow filtration.

[0142] In some embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 1 E7 TU/mL. In some embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 2E7, at least 3E7, at least 4E7, at least 5E7, at least 6E7, at least 7E7, at least 8E7, at least 9E7, or at least 1 E8 TU/mL. In certain embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 2E7 TU/mL. In some embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 3E7 TU/mL. In other embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 4E7 TU/mL. In yet other embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 5E7 TU/mL. In still other embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 6E7 TU/mL. In some embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 7E7 TU/mL. In certain embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 8E7 TU/mL. In other embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 9E7 TU/mL. In some embodiments, the lentiviral vector systems described herein result in a crude particle titer of at least 1 E8 TU/mL.

[0143] In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 500. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 450. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 400. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 350. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 300. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 250. In some embodiments, the lentiviral vector systems described herein result in a particle to infectivity ratio (P:l) at most 200.

[0144] In some embodiments, the present disclosure provides a lentiviral particle produced by the methods described herein.

[0145] In some embodiments, the present disclosure provides a method of expressing a GOI in a cell comprising contacting the cell with the composition of lentivirus particles. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.

[0146] In some embodiments, the present disclosure provides a method of expressing a GOI, comprising (i) introducing a lentiviral vector system described herein into a first cell; (ii) culturing the first cell under conditions where lentivirus particles are produced; (iii) harvesting the lentivirus particles from the culture supernatant; (iv) contacting a second cell with the lentivirus particles; and (v) expressing the GOI. In some embodiments, the second cell is in vitro. In some embodiments, the second cell is in vivo.

SEQUENCES:

FURTHER NUMBERED EMBODIMENTS

1 . A lentiviral transfer vector comprising: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four nucleotides at the 5’ end of the A 5’ LTR consist of GGGX, wherein X is A, T, or C; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

2. The lentiviral transfer vector of embodiment 1 , further comprising a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence comprising a truncated Gag gene (AGag); a polynucleotide sequence comprising a truncated Envelope gene (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; and a polypurine tract (PPT) polynucleotide sequence.

3. The lentiviral transfer vector of embodiment 1 or 2, wherein the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR.

4. The lentiviral transfer vector of embodiment 1 or 2, wherein the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR.

5. The lentiviral transfer vector of embodiment 3 or 4, wherein the HIV is HIV-1 subtype B.

6. The lentiviral transfer vector of embodiment 1 or 2, wherein the 3’ LTR is a self-inactivating 3’ LTR (SIN 3’ LTR).

7. The lentiviral transfer vector of embodiment 6, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

8. The lentiviral transfer vector of embodiment 7, wherein the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

9. The lentiviral transfer vector of embodiment 7 or 8, wherein the HIV is HIV-1 subtype B. 10. The lentiviral transfer vector of any one of embodiments 1 -9, wherein the A 5’ LTR and 3’ LTR are from the same HIV subtype.

11 . The lentiviral transfer vector of any one of embodiments 1-10, wherein the pA is a bovine growth hormone pA (bGH-pA).

12. The lentiviral transfer vector of any one of embodiments 1-11 , wherein the location for inserting the expression cassette is a multiple cloning site (MCS).

13 A lentiviral transfer vector, comprising, from 5’ to 3’: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv) wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

14. The lentiviral transfer vector of embodiment 13, further comprising a splice donor site and a splice acceptor site.

15. The lentiviral transfer vector of embodiment 14, wherein the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence.

16. The lentiviral transfer vector of embodiment 14 or 15, wherein the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence.

17. The lentiviral transfer vector of any one of embodiments 13-16, wherein the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR.

18. The lentiviral transfer vector of any one of embodiments 13-16, wherein the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR. 19. The lentiviral transfer vector of embodiment 17 or 18, wherein the HIV is HIV-1 subtype B.

20. The lentiviral transfer vector of any one of embodiments 13-16, wherein the 3’ LTR is a selfinactivating 3’ LTR (SIN 3’ LTR).

21 . The lentiviral transfer vector of embodiment 20, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

22. The lentiviral transfer vector of embodiment 21 , the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

23. The lentiviral transfer vector of embodiment 21 or 22, wherein the HIV is HIV-1 subtype B.

24. The lentiviral transfer vector of any one of embodiments 13-23, wherein the A 5’ LTR and 3’ LTR are from the same HIV subtype.

25. The lentiviral transfer vector of any one of embodiments 13-24, wherein the pA is a bovine growth hormone pA (bGH-pA).

26. The lentiviral transfer vector of any one of embodiments 13-25, wherein the location for inserting the expression cassette is a multiple cloning site (MCS).

27. The lentiviral transfer vector of any one of embodiments 1-26, wherein the CMV promoter lacking the TSS G comprises the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 1 .

28. The lentiviral transfer vector of any one of embodiments 1-26, wherein the A5’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:2 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 2.

29. The lentiviral transfer vector of any one of embodiments 1-26, wherein the 3’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 3.

30. The lentiviral transfer vector of any one of embodiments 1-26, wherein the pA polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:4 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 4.

31. The lentiviral transfer vector of any one of embodiments 2-26, wherein the Psi (T) viral packaging signal polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:5 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 5.

32. The lentiviral transfer vector of any one of embodiments 2-26, wherein the AGag polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:6 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 6.

33. The lentiviral transfer vector of any one of embodiments 2-26, wherein the AEnv polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NOT or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 7.

34. The lentiviral transfer vector of any one of embodiments 2-26, wherein the central polypurine tract (cPPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:8 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 8.

35. The lentiviral transfer vector of any one of embodiments 2-26, wherein the polypurine tract (PPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:9 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 9.

36. A lentiviral transfer vector, comprising the polynucleotide sequence of SEQ ID NO:10 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 10.

37. A lentiviral transfer vector of any one of embodiments 2-31 , 34, or 35, wherein the AGag-AEnv- RRE comprises the polynucleotide sequence of 15 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 15.

38. A lentiviral transfer vector of any one of embodiments 2-31 , 34, or 35, wherein the AGag-AEnv- RRE comprises the polynucleotide sequence of 17 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 17.

39. A lentiviral transfer vector of any one of embodiments 2-31 , 34, or 35, wherein the AGag-AEnv- RRE comprises the polynucleotide sequence of 19 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 19.

40. A lentiviral packaging vector comprising, from 5’ to 3’,

(i) an RSV promoter polynucleotide sequence;

(ii) a polynucleotide sequence encoding a Rev protein; and

(iii) an HSV thymidine kinase polyadenylation polynucleotide sequence.

41 . The lentiviral packaging vector of embodiment 40, comprising the polynucleotide sequence of SEQ ID NO:11.

42. A lentiviral packaging vector comprising, from 5’ to 3’,

(i) a CMV promoter polynucleotide sequence;

(ii) a human p-globin intron polynucleotide sequence;

(iii) a polynucleotide sequence encoding a Gag protein, a Pol protein, and a Rev response element (RRE); and

(iv) a rabbit p-globin polyadenylation polynucleotide sequence.

43. The lentiviral packaging vector of embodiment 42, comprising the polynucleotide sequence of SEQ ID NO:12.

44. A lentiviral packaging vector comprising, from 5’ to 3’:

(i) a CMV promoter polynucleotide sequence;

(ii) a rabbit p-globin intron polynucleotide sequence;

(iii) a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and

(iv) a human p-globin polyadenylation polynucleotide sequence.

45. The lentiviral packaging vector of embodiment 44, comprising the polynucleotide sequence of SEQ ID NO:13.

46. A lentiviral vector system comprising:

(a) a first packaging vector comprising a polynucleotide sequence encoding a Rev protein;

(b) a second packaging vector comprising a polynucleotide sequence encoding a Gag protein and a Pol protein; (c) a third packaging vector comprising a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and

(d) a transfer vector comprising, from 5’ to 3’: a cytomegalovirus (CMV) promoter polynucleotide sequence lacking the transcription start site (TSS) G; a truncated 5’ long terminal repeat (A5’ LTR) polynucleotide sequence located immediately downstream of the CMV promoter, wherein the first four-nucleotide sequence at the 5’ end of the A 5’ LTR consists of GGGX, wherein X is A, T, or C; a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence encoding a truncated Gag protein (AGag); a polynucleotide sequence encoding a truncated Envelope protein (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; a location for inserting an expression cassette comprising a polynucleotide sequence encoding a gene of interest (GOI); a polypurine tract (PPT) polynucleotide sequence; a 3’ long terminal repeat (3’ LTR) polynucleotide sequence; and a polyadenylation (pA) signal polynucleotide sequence.

47. The lentiviral vector system of embodiment 46, wherein the transfer vector further comprises a splice donor site and a splice acceptor site.

48. The lentiviral vector system of embodiment 47, wherein the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence.

49. The lentiviral vector system of embodiment 47 or 48, wherein the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence.

50. The lentiviral vector system of any one of embodiments 46-49, wherein the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR.

51 . The lentiviral vector system of any one of embodiments 46-49, wherein the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR.

52. The lentiviral vector system of embodiment 50 or 51 , wherein the HIV is HIV-1 subtype B.

53. The lentiviral vector system of any one of embodiments 46-49, wherein the 3’ LTR is a selfinactivating 3’ LTR (SIN 3’ LTR). 54. The lentiviral vector system of embodiment 53, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

55. The lentiviral vector system of embodiment 54, wherein the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

56. The lentiviral vector system of embodiment 53 or 54, wherein the HIV is HIV-1 subtype B.

57. The lentiviral vector system of any one of embodiments 46-56, wherein the A 5’ LTR and 3’ LTR are from the same HIV subtype.

58. The lentiviral vector system of any one of embodiments 46-57, wherein the pA is a bovine growth hormone pA (bGH-pA).

59. The lentiviral vector system of any one of embodiments 46-58, wherein the location for inserting the expression cassette is a multiple cloning site (MCS).

60. The lentiviral vector system of any one of embodiments 46-59, wherein the CMV promoter lacking the TSS G comprises the nucleic acid sequence of SEQ ID NO:1 .

61. The lentiviral vector system of any one of embodiments 46-59, wherein the A5’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:2.

62. The lentiviral vector system of any one of embodiments 46-59, wherein the 3’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:3.

63. The lentiviral vector system of any one of embodiments 46-59, wherein the pA polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:4.

64. The lentiviral vector system of any one of embodiments 46-59, wherein the Psi (T) viral packaging signal polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:5.

65. The lentiviral vector system of any one of embodiments 46-59, wherein the AGag polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:6.

66. The lentiviral vector system of any one of embodiments 46-59, wherein the AEnv polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NOT.

67. The lentiviral vector system of any one of embodiments 46-59, wherein the central polypurine tract (cPPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:8. 68. The lentiviral vector system of any one of embodiments 46-59, wherein the polypurine tract (PPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:9.

69. The lentiviral vector system of any one of embodiments 46-59, wherein the transfer vector comprises the polynucleotide sequence of SEQ ID NO:10.

70. The lentiviral vector system of any one of embodiments 46-69, wherein the first packaging vector comprises a polynucleotide sequence of SEQ ID NO:11 .

71 . The lentiviral vector system of any one of embodiments 46-70, wherein the second packaging vector comprises a polynucleotide sequence of SEQ ID NO:12.

72. The lentiviral vector system of any one of embodiments 46-71 , wherein the third packaging vector comprises a polynucleotide sequence of SEQ ID NO:13.

73. A lentiviral vector system, comprising:

(i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10;

(ii) a first packaging vector comprising a polynucleotide sequence of SEQ ID NO:11 ;

(iii) a second packaging vector comprising a polynucleotide sequence of SEQ ID NO:12; and

(iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13.

74. The lentiviral vector system of any one of embodiments 46-73, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL.

75. The lentiviral vector system of any one of embodiments 46-74, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E8 TU/mL.

76. The lentiviral vector system of any one of embodiments 46-75, wherein the lentiviral vector system is able to generate a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200.

77. The lentiviral vector system of any one of embodiments 46-76, wherein the lentiviral vector system is able to generate a P:l of at most 200.

78. The lentiviral transfer vector of any one of embodiments 1 -39, further comprising an expression cassette comprising a polynucleotide encoding the GOI. 79. The lentiviral transfer vector of embodiment 78, wherein the expression cassette comprises an EF1a promoter polynucleotide sequence.

80. The lentiviral transfer vector of embodiment 78, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide.

81 . The lentivirus transfer vector of embodiment 80, wherein the woodchuck hepatitis virus post- transcriptional regulatory element is a woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or a woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide.

82. The lentivirus transfer vector of any one of embodiments 80 or81 , wherein the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39.

83. The lentiviral transfer vector of embodiment 78, wherein the expression cassette comprises from 5’ to 3’:

(i) an EF1a promoter polynucleotide sequence;

(ii) a polynucleotide encoding the GOI; and

(iii) a woodchuck hepatitis virus post-transcriptional regulatory element variant 1 polynucleotide or a woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide.

84. The lentiviral transfer vector of embodiment 78, wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

85. The lentiviral vector system of any one of embodiments 46-77, further comprising an expression cassette comprising a polynucleotide encoding the GOI.

86. The lentiviral vector system of embodiment 85, wherein the expression cassette comprises an EF1a promoter polynucleotide sequence.

87. The lentiviral vector system of embodiment 85, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide.

88. The lentivirus vector system of embodiment 87, wherein the woodchuck hepatitis virus post- transcriptional regulatory element is a woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or a woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide.

89. The lentivirus vector system of any one of embodiments 87 or 88, wherein the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39.

90. The lentiviral vector system of embodiment 78, wherein the expression cassette comprises from 5’ to 3’:

(i) an EF1a promoter polynucleotide sequence;

(ii) a polynucleotide encoding the GOI; and

(iii) woodchuck hepatitis virus post-transcriptional regulatory element variant 1 or variant 2 polynucleotide.

91 . The lentiviral vector system of embodiment 78, wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

92. A method of producing a lentivirus, comprising:

(i) introducing the lentiviral vector system of any one of embodiments 46-77 or 85-91 into a cell;

(ii) culturing the cell under conditions where a lentivirus is produced; and

(iii) harvesting the lentivirus from the culture supernatant.

93. A lentivirus produced by the method of embodiment 92.

94. A method of expressing a GOI, comprising

(i) inserting an expression cassette comprising a polynucleotide sequence encoding the GOI in the lentiviral transfer vector of the lentiviral vector system of any one of embodiments 46-77;

(ii) introducing the lentiviral vector system into a first cell;

(iii) culturing the first cell under conditions where a lentivirus is produced;

(iv) harvesting the lentivirus from the culture supernatant;

(v) contacting a second cell with the lentivirus; and

(vi) expressing the GOI.

95. The method of embodiment 94, wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system. 96. A method of expressing a GOI, comprising

(i) introducing the lentiviral vector system of any one of embodiments 85-91 into a first cell;

(ii) culturing the first cell under conditions where a lentivirus is produced;

(iii) harvesting the lentivirus from the culture supernatant;

(iv) contacting a second cell with the lentivirus; and

(v) expressing the GOI.

97. The method of any one of embodiments 94-96, wherein the second cell is in vitro.

98. The method of any one of embodiments 94-96, wherein the second cell is in vivo.

99. A kit comprising a lentiviral vector system; wherein the lentiviral vector system comprises:

(b) a second packaging vector comprising a polynucleotide sequence encoding a Gag protein and a Pol protein;

(c) a third packaging vector comprising a polynucleotide sequence encoding a vesicular stomatitis virus (VSV) protein G (VSV-G); and

100. The kit of embodiment 99, wherein the transfer vector of the lentiviral vector system further comprises a splice donor site and a splice acceptor site.

101. The kit of embodiment 100, wherein the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence. 102. The kit of embodiment 100 or 101 , wherein the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence.

103. The kit of any one of embodiments 99-102, wherein the A5’ LTR of the transfer vector of the lentiviral vector system comprises an R region and a U5 region of an HIV 5’ LTR.

104. The kit of any one of embodiments 99-102, wherein the A5’ LTR of the transfer vector of the lentiviral vector system consists of an R region and a U5 region of an HIV 5’ LTR.

105. The kit of embodiment 103 or 104, wherein the HIV is HIV-1 subtype B.

106. The kit of any one of embodiments 99-102, wherein the 3’ LTR of the transfer vector of the lentiviral vector system is a self-inactivating 3’ LTR (SIN 3’ LTR).

107. The kit of embodiment 106, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

108. The kit of embodiment 107, wherein the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

109. The kit of embodiment 106 or 107, wherein the HIV is HIV-1 subtype B.

110. The kit of any one of embodiments 99-109, wherein the A 5’ LTR and 3’ LTR of the transfer vector of the lentiviral vector system are from the same HIV subtype.

111. The kit of any one of embodiments 99-110, wherein the pA of the transfer vector of the lentiviral vector system is a bovine growth hormone pA (bGH-pA).

112. The kit of any one of embodiments 99-111 , wherein the location for inserting the expression cassette of the transfer vector of the lentiviral vector system is a multiple cloning site (MCS).

113. The kit of any one of embodiments 99-112, wherein the CMV promoter lacking the TSS Gof the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:1 .

114. The kit of any one of embodiments 99-113, wherein the A5’ LTR polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:2.

115. The kit of any one of embodiments 99-114, wherein the 3’ LTR polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:3. 116. The kit of any one of embodiments 99-1 15, wherein the pA polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:4.

117. The kit of any one of embodiments 99-116, wherein the Psi (T) viral packaging signal polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:5.

118. The kit of any one of embodiments 99-117, wherein the AGag polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:6.

119. The kit of any one of embodiments 99-118, wherein the AEnv polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NOT.

120. The kit of any one of embodiments 99-119, wherein the central polypurine tract (cPPT) polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:8.

121. The kit of any one of embodiments 99-120, wherein the polypurine tract (PPT) polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:9.

122. The kit of any one of embodiments 99-121 , wherein the transfer vector of the lentiviral vector system comprises the polynucleotide sequence of SEQ ID NO: 10.

123. The kit of any one of embodiments 99-122, wherein the first packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:11.

124. The kit of any one of embodiments 99-123, wherein the second packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:12.

125. The kit of any one of embodiments 99-124, wherein the third packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:13.

126. A kit comprising a lentiviral vector system, comprising:

(i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10;

(iv) a third packaging vector comprising a polynucleotide sequence of SEQ ID NO:13. 127. The kit of any one of embodiments 99-126, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL.

128. The kit of any one of embodiments 99-124, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E8 TU/mL.

129. The kit of any one of embodiments 99-128, wherein the lentiviral vector system is able to generate a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200.

130. The kit of any one of embodiments 99-129, wherein the lentiviral vector system is able to generate a P:l of at most 200.

131. The kit of any one of embodiments 99-130, wherein the transfer vector further comprisies an expression cassette comprising a polynucleotide encoding the GOI.

132. The kit of embodiment 131 , wherein the expression cassette comprises an EF1a promoter polynucleotide sequence.

133. The kit of embodiment 131 , wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide.

134. The kit of embodiment 133, wherein the woodchuck hepatitis virus post-transcriptional regulatory element is woodchuck hepatitis virus post transcriptional regulatory element variant 1 or variant 2 polynucleotide.

135. The kit of any one of embodiments 133 or 134, wherein the woodchuck hepatitis virus post- transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39.

136. The kit of embodiment 131 , wherein the expression cassette comprises from 5’ to 3’:

(i) an EF1a promoter polynucleotide sequence;

(ii) a polynucleotide encoding the GOI; and

(iii) a woodchuck hepatitis virus post-transcriptional regulatory element variant 1 or variant 2 polynucleotide.

137. The kit of embodiment 131 , wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system. EXAMPLES

Example 1: Optimization of AGag- AEnv-RRE sequence in a lentiviral transfer vector

[0147] Experiments were performed to optimize the AGag-AEnv-RRE sequence in a lentiviral transfer vector. (Fig. 3). The optimized AGag-AEnv-RRE sequences were designed to improve crude titer and decrease the P:l ratio. Nine different variants of the AGag- AEnv-RRE sequence were evaluated (see SEQ ID NOS: 14-22).

[0148] Fig. 4A shows that all nine of the lentiviral transfer vectors comprising the variant AGag- AEnv- RRE sequences had titers of > 10⁷ TU/ mL. Fig. 4B shows that pEB-00500, pEB-00509, and pEB- 00511 demonstrated the highest p24 levels (Fig. 4B) and the lowest P:l ratio (Fig. 4C). The AGag- AEnv-RRE sequences of pEB-00500, pEB-00509, and pEB-00511 were selected for further development. The AGag- AEnv-RRE sequence of pEB-00500 was transferred into “Transfer Vector #1.” The AGag- AEnv-RRE sequence of pEB-00509 was transferred into “Transfer Vector #2.” The AGag- AEnv-RRE sequence of pEB-00511 was transferred into “Transfer Vector #3.”

Example 2: Optimization of Lentiviral Packaging Vectors

[0149] Experiments were performed to optimize the combination of lentiviral packaging vectors. Lentivirus was produced using the Transfer Vector #1 and a combination of packaging plasmids from the table below. Transfer Vector #1 contains a CD19 CAR as the GOI. Fig. 5A shows the Rev Packaging Vectors, pEB-00516 and pEB-00517. Fig. 5B shows the Gag-Pol Packaging Vectors, pEB- 00520 and pEB-00521. Fig. 5C shows the VSV-G Packaging Vectors, pEB-00518 and pEB-00519.

An X indicates that the packaging vector was used. [0150] Fig. 6A shows that the crude lentiviral titers of the lentiviral transfect vector and packaging plasmids described herein were superior to the crude titer of lentivirus produced with commercially available packaging plasmids. Fig. 6B shows the p24 levels and Fig. 6C shows the P:l ratios for each combination. Combination 2 and Combination 6 were selected for further experiments because these two combinations had the best combination of qPCR titer, p24 titer, and P:l ratio. “Commercial” in Figs. 6A-6C refers to a control in which commercial packaging plasmids and a commercial transfer vector were utilized. “Commercial Pack” in Figs. 6A-C refers to a control in which commercial packaging plasmids and the EB-LVV transfer vector (pEB-00515) were utilized.

[0151] Lentiviral systems comprising Combination 2 packaging plasmids, Combination 6 packaging plasmids, or commercial packaging plasmids were evaluated, (see Table below). Fig. 7A shows the crude lentiviral titers of each combination of lentiviral packaging plasmids and lentiviral transfer vectors. The use of Combination 2 and Combination 6 packaging plasmids in combination with the lentiviral transfer vector pEB-00515 achieved higher viral titers than commercial packaging plasmids. Fig. 7B shows the p24 titers for each combination, and Fig. 7C shows the P:l ratio for each combination. The Combination 6 packaging plasmids resulted in higher p24 titers than Combination 2 packaging plasmids. Both Combination 2 packaging plasmids and Combination 6 packaging plasmids had a P:l ratio that was superior to (i.e., lower) than the P:l ratio obtained with commercial packaging plasmids. Combination 6 packaging plasmids were selected for future development.

[0152] Lentivirus production using a lentiviral system containing Combination 6 packaging plasmids and lentiviral Transfer Vectors #1 , #2, or #3 were evaluated. Lentiviral Transfer Vector #1 contained a AGag- AEnv-RRE sequence identical to that of pEB-00500. Lentiviral Transfer Vector #2 contained a AGag- AEnv-RRE sequence identical to that of pEB-00509. Lentiviral Transfer Vector #3 contained a AGag- AEnv-RRE sequence identical to that of pEB-00511 . The crude lentiviral titers, p24 titers, and P:l ratio was evaluated when the GOI for lentiviral Transfer Vectors #1 , #2, and #3 was a CD19 chimeric antigen receptor (CAR, Figs. 8A-8C), a MAGEA1-T cell Receptor (TCR, Figs. 9A-9C), or a Flt3 CAR (Figs. 10A-10C). Transfer Vector #3 and Transfer Vector #1 produced crude lentiviral titers (Fig. 8A) and P:l ratios (Fig. 8C) superior to Transfer Vector #2 when the GOI was CD19 CAR. Transfer Vector #3 produced crude lentiviral titers (Fig. 9A) and a P:l ratio that were superior to Transfer Vector #2 and Transfer Vector #1 when the GOI was MAGEA1-TCR (Fig. 9C).Transfer Vector #3 produced crude lentiviral titers (Fig. 10A) and a P:l ratio that were superior to Transfer Vector #2 when the GOI was Flt3 CAR (Fig. 10C). Transfer Vector #3 was selected for future studies. Transfer Vector #3 results in superior lentiviral titers to Transfer Vector #2 when the GOI is CD19 CAR (Fig. 11A), MAGEA1-TCR (Fig. 11B), or Flt3 CAR (Fig. 11C). Transfer Vector #3 results in superior lentiviral titers to Transfer Vector #1 when the GOI is MAGEA1-TCR (Fig. 11B) or Flt3 CAR (Fig. 11C).

Example 3: Optimization of Lentiviral Rev Packaging Vector

[0153] Experiments were performed to optimize the polyA sequence in the Rev lentiviral packaging plasmid. The pEB-00516, pEB-00537, and pEB-00538 Rev packaging plasmids were evaluated in lentiviral systems containing a pEB-00521 Gag/Pol packaging plasmid, a pEB-00519 VSVG packaging plasmid, and a pEB-00536 lentiviral transfer vector. The pEB-00536 lentiviral transfer vector has the same AGag- AEnv-RRE sequence as that of pEB-00511 . The pEB-00516 Rev packaging plasmid contained the HIV-1 polyA sequence. The pEB-00537 Rev packaging plasmid contained a synthetic polyA sequence. The pEB-00538 Rev packaging plasmid contained a HSV-tk polyA sequence. Commercial Rev packaging plasmids were used as a control. Lentivirus expressing GFP were used as an internal control to validate titers.

[0154] Lentiviral systems containing the pEB-00538 Rev packaging plasmid, which had the HSV-tk polyA sequence exhibited the highest crude viral titers. Lentiviral systems containing the pEB-00538 Rev packaging plasmid; pEB-00521 Gag/Pol packaging plasmid; and pEB-00519 VSVG packaging plasmid were selected for future experiments. (Fig. 12). The lentivirus production conditions of Fig. 12 (#1 , #2, #3, #4, #5) are described in the table below.

Example 4: Optimization of Lentiviral Transduction Conditions

[0155] Experiments were performed to optimize the amount of sodium butyrate and molar ratio of the plasmids used in the lentiviral systems. The table below shows the conditions that were evaluated. Lentivirus expressing GFP were used as an internal transduction control.

*The plasmid molar ratio represents the ratio of lentiviral transfer vector: Gag/Pol Packaging Vector: Rev Packaging Vector; VSV-G Packaging Vector.

[0156] As a control, two conditions were tested: “536-LV-Max” and “1101-LV-Max.”

[0157] The 536-LV-Max lentiviral system utilized 15.5 pg of the pEB-00521 Gag/Pol Packaging Vector; 11 .7 pg of the pEB-00516 Rev Packaging Vector; and 5 pg of the pEB-00519 VSV-G Packaging Vector; and 27.7 pg of the pEB-00536 Lentiviral Transfer Vector. The plasmid molar ratio of lentiviral transfer vector: Gag/Pol Packaging Vector: Rev Packaging Vector; VSV-G Packaging Vector was 3.8: 2: 4: 1.

[0158] The 1101-LV-Max lentiviral system utilized 22.5 pg of the pEB-00401 Gag/Pol Packaging Vector; 15 pg of the pEB-00400 Rev Packaging Vector; and 5 pg of the pEB-00402 VSV-G Packaging Vector; and 30 pg of the pEB-01101 Lentiviral Transfer Vector. The plasmid molar ratio of lentiviral transfer vector: Gag/Pol Packaging Vector: Rev Packaging Vector; VSV-G Packaging Vector was: 3.2:2.1 :3.9:1 .

[0159] Conditions 3, 4, and 11 each resulted in lentiviral titers > 10⁷ TU/mL (Fig. 13A). Fig. 13B shows the p24 titer for each condition, and Fig. 13C shows the P:l ratio for each condition. Condition 11 was selected for future optimization because it had the lowest P:l ratio.

Example 5: Optimization of the sequence at the junction of CMV promoter and A5’ LTR in the lentiviral transfer vector

[0160] Experiments were performed to assess the optimization of the sequence at the junction of the CMV promoter and the A5’ LTR in the lentiviral transfer vector, designed to improve crude titer and decrease the P:l ratio. Optimization strategies are outlined in Fig. 14. Briefly, four variations of the CMV promoter and A5’ LTR were made to alter the sequence at the junction of the transcriptional start site(TSS) G and the A5’ LTR: (a) pEB-00536 - comprises a CMV promoter sequence with the TSS G and a A5’ LTR comprising 3 G’s at the 5’ terminus, wherein a BsiW1 restriction site is inserted between the CMV promoter sequence and the A5’ LTR. pEB-00536 functions as a control vector in these experiments;

(b) pEB-00539 - comprises a CMV promoter sequence that lacks the TSS G and a modified A5’ LTR comprising 1 G at the 5’ terminus. pEB-00539 does not comprise a BsiW1 restriction site;

(c) pEB-00540 - comprises a CMV promoter sequence with the TSS G and a modified A5’ LTR lacking any G’s at the 5’ terminus, wherein a BsiW1 restriction site is inserted between the CMV promoter sequence and the modified A5’ LTR; and

(d) pEB-00541 - comprises a CMV promoter sequence that lacks the TSS G and a A5’ LTR comprising 3 G’s at the 5’ terminus. pEB-00541 does not comprise a BsiW1 restriction site.

[0161] The above transfer vector was used in combination with the same three packaging vectors: pEB-00519, pEB-00521 , and pEB-00538. Lentivirus was produced used condition #11 of Example 4. Lentivirus producer cells were transfected with the packaging vectors and transfer vector. 16 hours later sodium butyrate was added to the cell culture. 48 hours after transfection, lentivirus was harvested from the cell culture media.

[0162] Functional results achieved with each of these modifications are shown in Fig. 15A - Fig. 15C.

[0163] As shown in Fig. 15A, reducing the triple G in the A5’ LTR to a single G lowered overall titers (See pEB-00536 vs pEB-00540; and pEB-00541 vs pEB-00539). Further, removing the BsiWI site in the construct improved crude titers 3-5 fold (See pEB-00536 vs pEB-00541 and pEB-00540 vs pEB- 00539). Crude titers of 1.36E8 TU/mL were achieved using pEB-00541 which comprises a CMV promoter lacking the TSS G and immediately downstream triple Gs of the A5’ LTR. pEB-00541 also demonstrated the highest p24 levels (Fig. 15B) and the lowest P:l ratio (Fig. 15C).

Example 6: Comparison of the current lentiviral vector system to a commercially available lentiviral vector system

[0164] Experiments were performed to compare the lentiviral systems described herein with a commercially available system, wherein the commercially available lentiviral transfer vector comprises a CMV promoter including the TSS G and immediately downstream triple Gs of the A5’ LTR. Two different genes of interest (a Flt3 CAR or GFP) were inserted into the pEB-00541 transfer vector described in Example 5 or the commercially available transfer vector. Viral titers and P:l ratios were determined for each transfer vector. As shown in Fig. 16A and Fig. 16B for Flt3 CAR and Fig. 17A and Fig. 17B for GFP, the current transfer vector design achieved increased viral titers and decreased P:l ratios compared to the commercial vector. INCORPORATION BY REFERENCE

[0165] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

2. The lentiviral transfer vector of claim 1 , further comprising a Psi (T) viral packaging signal polynucleotide sequence; a polynucleotide sequence comprising a truncated Gag gene (AGag); a polynucleotide sequence comprising a truncated Envelope gene (AEnv), wherein the AEnv comprises a Rev response element (RRE); a central polypurine tract (cPPT) polynucleotide sequence; and a polypurine tract (PPT) polynucleotide sequence.

3. The lentiviral transfer vector of claim 1 or 2, wherein the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR.

4. The lentiviral transfer vector of claim 1 or 2, wherein the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR.

5. The lentiviral transfer vector of claim 3 or 4, wherein the HIV is HIV-1 subtype B.

6. The lentiviral transfer vector of claim 1 or 2, wherein the 3’ LTR is a self-inactivating 3’ LTR (SIN 3’ LTR).

7. The lentiviral transfer vector of claim 6, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

8. The lentiviral transfer vector of claim 7, wherein the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

9. The lentiviral transfer vector of claim 7 or 8, wherein the HIV is HIV-1 subtype B.

10. The lentiviral transfer vector of any one of claims 1 -9, wherein the A 5’ LTR and 3’ LTR are from the same HIV subtype.

11. The lentiviral transfer vector of any one of claims 1-10, wherein the pA is a bovine growth hormone pA (bGH-pA).

12. The lentiviral transfer vector of any one of claims 1-11 , wherein the location for inserting the expression cassette is a multiple cloning site (MCS).

14. The lentiviral transfer vector of claim 13, further comprising a splice donor site and a splice acceptor site.

15. The lentiviral transfer vector of claim 14, wherein the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence.

16. The lentiviral transfer vector of claim 14 or 15, wherein the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence.

17. The lentiviral transfer vector of any one of claims 13-16, wherein the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR.

18. The lentiviral transfer vector of any one of claims 13-16, wherein the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR.

19. The lentiviral transfer vector of claim 17 or 18, wherein the HIV is HIV-1 subtype B.

20. The lentiviral transfer vector of any one of claims 13-16, wherein the 3’ LTR is a self-inactivating 3’ LTR (SIN 3’ LTR).

21 . The lentiviral transfer vector of claim 20, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

22. The lentiviral transfer vector of claim 21 , the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

23. The lentiviral transfer vector of claim 21 or 22, wherein the HIV is HIV-1 subtype B.

24. The lentiviral transfer vector of any one of claims 13-23, wherein the A 5’ LTR and 3’ LTR are from the same HIV subtype.

25. The lentiviral transfer vector of any one of claims 13-24, wherein the pA is a bovine growth hormone pA (bGH-pA).

26. The lentiviral transfer vector of any one of claims 13-25, wherein the location for inserting the expression cassette is a multiple cloning site (MCS).

27. The lentiviral transfer vector of any one of claims 1 -26, wherein the CMV promoter lacking the TSS G comprises the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 1 .

28. The lentiviral transfer vector of any one of claims 1-26, wherein the A5’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:2 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 2.

29. The lentiviral transfer vector of any one of claims 1-26, wherein the 3’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 3.

30. The lentiviral transfer vector of any one of claims 1 -26, wherein the pA polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:4 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 4.

31 . The lentiviral transfer vector of any one of claims 2-26, wherein the Psi (T) viral packaging signal polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:5 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 5.

32. The lentiviral transfer vector of any one of claims 2-26, wherein the AGag polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:6 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 6.

33. The lentiviral transfer vector of any one of claims 2-26, wherein the AEnv polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NOT or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 7.

34. The lentiviral transfer vector of any one of claims 2-26, wherein the central polypurine tract (cPPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:8 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 8.

35. The lentiviral transfer vector of any one of claims 2-26, wherein the polypurine tract (PPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:9 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 9.

37. A lentiviral transfer vector of any one of claims 2-31 , 34, or 35, wherein the AGag-AEnv-RRE comprises the polynucleotide sequence of 15 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 15.

38. A lentiviral transfer vector of any one of claims 2-31 , 34, or 35, wherein the AGag-AEnv-RRE comprises the polynucleotide sequence of 17 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 17.

39. A lentiviral transfer vector of any one of claims 2-31 , 34, or 35, wherein the AGag-AEnv-RRE comprises the polynucleotide sequence of 19 or a nucleic acid with at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, or at least about 99 % identity to the nucleic acid of SEQ ID NO: 19.

40. A lentiviral packaging vector comprising, from 5’ to 3’,

(i) an RSV promoter polynucleotide sequence;

(ii) a polynucleotide sequence encoding a Rev protein; and

(iii) an HSV thymidine kinase polyadenylation polynucleotide sequence.

41 . The lentiviral packaging vector of claim 40, comprising the polynucleotide sequence of SEQ ID NO:11.

42. A lentiviral packaging vector comprising, from 5’ to 3’,

(i) a CMV promoter polynucleotide sequence;

(ii) a human p-globin intron polynucleotide sequence;

(iv) a rabbit p-globin polyadenylation polynucleotide sequence.

43. The lentiviral packaging vector of claim 42, comprising the polynucleotide sequence of SEQ ID NO:12.

44. A lentiviral packaging vector comprising, from 5’ to 3’:

(i) a CMV promoter polynucleotide sequence;

(ii) a rabbit p-globin intron polynucleotide sequence;

(iv) a human p-globin polyadenylation polynucleotide sequence.

45. The lentiviral packaging vector of claim 44, comprising the polynucleotide sequence of SEQ ID NO:13.

46. A lentiviral vector system comprising:

47. The lentiviral vector system of claim 46, wherein the transfer vector further comprises a splice donor site and a splice acceptor site.

48. The lentiviral vector system of claim 47, wherein the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence.

49. The lentiviral vector system of claim 47 or 48, wherein the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence.

50. The lentiviral vector system of any one of claims 46-49, wherein the A5’ LTR comprises an R region and a U5 region of an HIV 5’ LTR.

51 . The lentiviral vector system of any one of claims 46-49, wherein the A5’ LTR consists of an R region and a U5 region of an HIV 5’ LTR.

52. The lentiviral vector system of claim 50 or 51 , wherein the HIV is HIV-1 subtype B.

53. The lentiviral vector system of any one of claims 46-49, wherein the 3’ LTR is a self-inactivating 3’ LTR (SIN 3’ LTR).

54. The lentiviral vector system of claim 53, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

55. The lentiviral vector system of claim 54, wherein the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

56. The lentiviral vector system of claim 53 or 54, wherein the HIV is HIV-1 subtype B.

57. The lentiviral vector system of any one of claims 46-56, wherein the A 5’ LTR and 3’ LTR are from the same HIV subtype.

58. The lentiviral vector system of any one of claims 46-57, wherein the pA is a bovine growth hormone pA (bGH-pA).

59. The lentiviral vector system of any one of claims 46-58, wherein the location for inserting the expression cassette is a multiple cloning site (MCS).

60. The lentiviral vector system of any one of claims 46-59, wherein the CMV promoter lacking the TSS G comprises the nucleic acid sequence of SEQ ID NO:1 .

61 . The lentiviral vector system of any one of claims 46-59, wherein the A5’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:2.

62. The lentiviral vector system of any one of claims 46-59, wherein the 3’ LTR polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:3.

63. The lentiviral vector system of any one of claims 46-59, wherein the pA polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:4.

64. The lentiviral vector system of any one of claims 46-59, wherein the Psi (T) viral packaging signal polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:5.

65. The lentiviral vector system of any one of claims 46-59, wherein the AGag polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:6.

66. The lentiviral vector system of any one of claims 46-59, wherein the AEnv polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NOT.

67. The lentiviral vector system of any one of claims 46-59, wherein the central polypurine tract (cPPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:8.

68. The lentiviral vector system of any one of claims 46-59, wherein the polypurine tract (PPT) polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO:9.

69. The lentiviral vector system of any one of claims 46-59, wherein the transfer vector comprises the polynucleotide sequence of SEQ ID NO:10.

70. The lentiviral vector system of any one of claims 46-69, wherein the first packaging vector comprises a polynucleotide sequence of SEQ ID NO:11 .

71 . The lentiviral vector system of any one of claims 46-70, wherein the second packaging vector comprises a polynucleotide sequence of SEQ ID NO:12.

72. The lentiviral vector system of any one of claims 46-71 , wherein the third packaging vector comprises a polynucleotide sequence of SEQ ID NO:13.

73. A lentiviral vector system, comprising:

(i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10;

74. The lentiviral vector system of any one of claims 46-73, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL.

75. The lentiviral vector system of any one of claims 46-74, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E8 TU/mL.

76. The lentiviral vector system of any one of claims 46-75, wherein the lentiviral vector system is able to generate a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200.

77. The lentiviral vector system of any one of claims 46-76, wherein the lentiviral vector system is able to generate a P:l of at most 200.

78. The lentiviral transfer vector of any one of claims 1-39, further comprising an expression cassette comprising a polynucleotide encoding the GOI.

79. The lentiviral transfer vector of claim 78, wherein the expression cassette comprises an EF1a promoter polynucleotide sequence.

80. The lentiviral transfer vector of claim 78, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide.

81. The lentivirus transfer vector of claim 80, wherein the woodchuck hepatitis virus post- transcriptional regulatory element is a woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or a woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide.

82. The lentivirus transfer vector of any one of claims 80 or81 , wherein the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39.

83. The lentiviral transfer vector of claim 78, wherein the expression cassette comprises from 5’ to 3’:

(i) an EF1 a promoter polynucleotide sequence;

(ii) a polynucleotide encoding the GOI; and

84. The lentiviral transfer vector of claim 78, wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

85. The lentiviral vector system of any one of claims 46-77, further comprising an expression cassette comprising a polynucleotide encoding the GOI.

86. The lentiviral vector system of claim 85, wherein the expression cassette comprises an EF1 a promoter polynucleotide sequence.

87. The lentiviral vector system of claim 85, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide.

88. The lentivirus vector system of claim 87, wherein the woodchuck hepatitis virus post- transcriptional regulatory element is a woodchuck hepatitis virus post transcriptional regulatory element variant 1 polynucleotide or a woodchuck hepatitis virus post transcriptional regulatory element variant 2 polynucleotide.

89. The lentivirus vector system of any one of claims 87 or 88, wherein the woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39.

90. The lentiviral vector system of claim 78, wherein the expression cassette comprises from 5’ to 3’:

(i) an EF1 a promoter polynucleotide sequence;

(ii) a polynucleotide encoding the GOI; and

91. The lentiviral vector system of claim 78, wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

92. A method of producing a lentivirus, comprising:

(i) introducing the lentiviral vector system of any one of claims 46-77 or 85-91 into a cell;

(ii) culturing the cell under conditions where a lentivirus is produced; and

(iii) harvesting the lentivirus from the culture supernatant.

93. A lentivirus produced by the method of claim 92.

94. A method of expressing a GOI, comprising

(i) inserting an expression cassette comprising a polynucleotide sequence encoding the GOI in the lentiviral transfer vector of the lentiviral vector system of any one of claims 46-77;

(ii) introducing the lentiviral vector system into a first cell;

(iii) culturing the first cell under conditions where a lentivirus is produced;

(iv) harvesting the lentivirus from the culture supernatant;

(v) contacting a second cell with the lentivirus; and

(vi) expressing the GOI.

95. The method of claim 94, wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.

96. A method of expressing a GOI, comprising

(i) introducing the lentiviral vector system of any one of claims 85-91 into a first cell;

(ii) culturing the first cell under conditions where a lentivirus is produced; (iii) harvesting the lentivirus from the culture supernatant;

(iv) contacting a second cell with the lentivirus; and

(v) expressing the GOI.

97. The method of any one of claims 94-96, wherein the second cell is in vitro.

98. The method of any one of claims 94-96, wherein the second cell is in vivo.

100. The kit of claim 99, wherein the transfer vector of the lentiviral vector system further comprises a splice donor site and a splice acceptor site.

101. The kit of claim 100, wherein the splice donor site is within the Psi (T) viral packaging signal polynucleotide sequence.

102. The kit of claim 100 or 101 , wherein the splice acceptor site is between the AEnv polynucleotide sequence and the cPPT polynucleotide sequence.

103. The kit of any one of claims 99-102, wherein the A5’ LTR of the transfer vector of the lentiviral vector system comprises an R region and a U5 region of an HIV 5’ LTR.

104. The kit of any one of claims 99-102, wherein the A5’ LTR of the transfer vector of the lentiviral vector system consists of an R region and a U5 region of an HIV 5’ LTR.

105. The kit of claim 103 or 104, wherein the HIV is HIV-1 subtype B.

106. The kit of any one of claims 99-102, wherein the 3’ LTR of the transfer vector of the lentiviral vector system is a self-inactivating 3’ LTR (SIN 3’ LTR).

107. The kit of claim 106, wherein the SIN 3’ LTR comprises an R region and a U5 region of an HIV 3’ LTR.

108. The kit of claim 107, wherein the SIN 3’ LTR further comprises a portion of a U3 region of the HIV 3’ LTR.

109. The kit of claim 106 or 107, wherein the HIV is HIV-1 subtype B.

110. The kit of any one of claims 99-109, wherein the A 5’ LTR and 3’ LTR of the transfer vector of the lentiviral vector system are from the same HIV subtype.

111. The kit of any one of claims 99-110, wherein the pA of the transfer vector of the lentiviral vector system is a bovine growth hormone pA (bGH-pA).

112. The kit of any one of claims 99-111 , wherein the location for inserting the expression cassette of the transfer vector of the lentiviral vector system is a multiple cloning site (MCS).

113. The kit of any one of claims 99-112, wherein the CMV promoter lacking the TSS Gof the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:1 .

114. The kit of any one of claims 99-113, wherein the A5’ LTR polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:2.

115. The kit of any one of claims 99-114, wherein the 3’ LTR polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:3.

116. The kit of any one of claims 99-115, wherein the pA polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:4.

117. The kit of any one of claims 99-116, wherein the Psi (T) viral packaging signal polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:5.

118. The kit of any one of claims 99-117, wherein the AGag polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:6.

119. The kit of any one of claims 99-118, wherein the AEnv polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:7.

120. The kit of any one of claims 99-119, wherein the central polypurine tract (cPPT) polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:8.

121. The kit of any one of claims 99-120, wherein the polypurine tract (PPT) polynucleotide sequence of the transfer vector of the lentiviral vector system comprises the nucleic acid sequence of SEQ ID NO:9.

122. The kit of any one of claims 99-121 , wherein the transfer vector of the lentiviral vector system comprises the polynucleotide sequence of SEQ ID NQ:10.

123. The kit of any one of claims 99-122, wherein the first packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:11 .

124. The kit of any one of claims 99-123, wherein the second packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:12.

125. The kit of any one of claims 99-124, wherein the third packaging vector of the lentiviral vector system comprises a polynucleotide sequence of SEQ ID NO:13.

126. A kit comprising a lentiviral vector system, comprising:

(i) a transfer vector comprising a polynucleotide sequence of SEQ ID NQ:10;

127. The kit of any one of claims 99-126, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E7, 2E7, 3E7, 4E7, 5E7, 6E7, 7E7, 8E7, 9E7, or 1 E8 TU/mL.

128. The kit of any one of claims 99-124, wherein the lentiviral vector system is able to generate a crude titer of at least 1 E8 TU/mL.

129. The kit of any one of claims 99-128, wherein the lentiviral vector system is able to generate a particle to infectivity ratio (P:l) of at most 500, 450, 400, 350, 300, 250, or 200.

130. The kit of any one of claims 99-129, wherein the lentiviral vector system is able to generate a P:l of at most 200.

131 . The kit of any one of claims 99-130, wherein the transfer vector further comprisies an expression cassette comprising a polynucleotide encoding the GOI.

132. The kit of claim 131 , wherein the expression cassette comprises an EF1a promoter polynucleotide sequence.

133. The kit of claim 131 , wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element polynucleotide.

134. The kit of claim 133, wherein the woodchuck hepatitis virus post-transcriptional regulatory element is woodchuck hepatitis virus post transcriptional regulatory element variant 1 or variant 2 polynucleotide.

135. The kit of any one of claims 133 or 134, wherein the woodchuck hepatitis virus post- transcriptional regulatory element polynucleotide comprises a sequence of any one of SEQ ID NOS: 37-39 or a sequence with at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 97 %, at least 98 %, at least 99 %, or at least 99.5 identical to any one of SEQ ID NOS: 37-39.

136. The kit of claim 131 , wherein the expression cassette comprises from 5’ to 3’:

(i) an EF1a promoter polynucleotide sequence;

(ii) a polynucleotide encoding the GOI; and

137. The kit of claim 131 , wherein the GOI is selected from a cytokine, a chemokine, an enzyme, an engineered T cell receptor, a chimeric antigen receptor (CAR), and a CRISPR/Cas system.