Methods and products for prime editing genetic engineering

Abstract

The present invention relates to the field of gene targeting by methods using viral-derived vector systems related to the Prime editing genetic engineering system Optimizing the delivery of prime editing tools via virus-like particles (VLPs) necessitates careful consideration and adaptation of the prime editing system to ensure efficient cargo packaging, delivery, and expression within target cells. The inventors were able to demonstrate that the molecular improvements of constructs of the Prime editing tool according to the invention enabled enhanced fidelity of editing in human and mammalian cells, such as iPSC. In particular, the present invention relates to a virus-derived particle (VLP) comprising a (i) fusion protein comprising a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS) and a prime editor.

Description

[0001] TITLE

[0002] METHODS AND PRODUCTS FOR PRIME EDITING GENETIC ENGINEERING

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of gene targeting by methods using viral-derived vector systems related to the Prime editing genetic engineering system.

[0005] BACKGROUND OF THE INVENTION

[0006] In recent decades, advancements in molecular biology have paved the way for revolutionary approaches in treating genetic diseases. Among these approaches, gene targeting has emerged as a promising avenue for precise manipulation of the genetic code to address a myriad of medical conditions. The ability to selectively edit genetic sequences holds immense potential for developing tailored therapies, thereby offering hope for patients afflicted with genetic disorders.

[0007] Despite the promising prospects of gene targeting, existing methodologies are fraught with challenges, necessitating continual innovation in the field of genetic engineering. The CRISPR / Cas9 system, hailed as a groundbreaking advancement in genome editing, has revolutionized molecular biology by offering unprecedented precision and efficiency in genetic manipulation. Its simplicity, versatility, and ability to target specific genomic loci with remarkable accuracy have catalyzed a wave of research and therapeutic development. However, like any pioneering technology, CRISPR / Cas9 is not immune to limitations. Concerns regarding off-target effects, unintended genetic modifications, and the potential for immune responses have been raised, highlighting the need for alternative strategies that can complement and augment its capabilities. These challenges, while significant, have spurred the development of innovative approaches such as Prime Editing (PE), which seek to address the shortcomings of existing gene editing tools while building upon their remarkable successes.

[0008] The advent of Prime Editing holds immense promise for revolutionizing the landscape of genetic medicine. By overcoming the limitations associated with existing gene editing tools, prime editing offers a transformative approach for the treatment of genetic diseases. Its ability to precisely rewrite genetic code without inducing double-strand breaks or relying on donor DNA templates makes it particularly well-suited for correcting a diverse array of genetic mutations with unparalleled efficiency and accuracy. Since pegRNA recognizes its target on both DNA strands at the level of the crRNA and at the level of the PBS, this double checkpoint limits the risk of illegitimate hybridizations. These reasons render Prime Editing far more precise than classical CRISPR CAS9 systems.

[0009] This enhanced precision holds great potential for the use of Prime Editing in molecular medecine and catalyses the global technological efforts to increment the PE-toolbox.

[0010] As documented by Anzalone et al. (Nature volume 576, pages 149-157 (2019)) by analysing pathological variants of the ClinVar database, 85-99% of genetic disorders in human (deletions, in dels, duplications) are <30 bp in-length which is the range of action of Prime Editing, making it particularly adapted to small edits. Prime Editing may correct up to 89% of the 75122 pathogenic human genetic variants. Nonetheless, safe administration of Prime Editing within human cells remains an essential and mandatory challenge.

[0011] Prime editing tools are commonly delivered to target cells via transfection methods, which involve the introduction of exogenous nucleic acids into cells to enable genome editing. Transfection can be achieved using various techniques, including lipid-mediated transfection, electroporation, nucleofection or plasmid transfection. Plasmid transfection involves the direct introduction of plasmid DNA containing the prime editing components into cells, which then undergo expression to mediate genome editing.

[0012] While these transfection methods offer high efficiency and versatility, they can also be associated with cytotoxicity, cellular stress, and reduced cell viability, particularly in primary cells or sensitive cell lines.

[0013] Alternative delivery methods, such as virus-derived particles (VLPs), offer an attractive solution to these drawbacks by providing efficient cargo delivery while minimizing cytotoxic effects, thereby enhancing the viability and applicability of genome editing technologies for various cellular contexts (W02017068077 Al).

[0014] One major advantage of RNP-delivering VLPs (ribonucleoprotein-delivering VLPs) is their capacity to transfer their cargo without introduction of coding DNA in recipient cells. As opposed to plasmid transfection, this obviously avoids the risk of DNA-integration into recipient cells and the amplitude of cell response induced by foreign-DNA. In addition, RNP-delivery mediated by VLP is transient while plasmid transfection can drive expression of active proteins for several days. Consequently, RNP-delivering VLPs induce less off-targets activities as compared with transfection which further increases their value for therapeutical purposes (Lyu, P. & Lu, B. New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery. Int. J. Mol. Sci. 23, 8750 (2022)).

[0015] Optimizing the delivery of prime editing tools via virus-like particles (VLPs) necessitates careful consideration and adaptation of the prime editing system to ensure efficient cargo packaging, delivery, and expression within target cells.

[0016] Therefore, there is a need in the art for improved tools and methods for gene editing by using prime editing technology.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention relates to a virus-derived particle comprising a fusion protein comprising: a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity;

[0019] In a particular embodiment, the virus-derived particle (VLP) further comprises at least one prime editing guide RNA (pegRNA), in particular a pegRNA bound to the napDNAbp.

[0020] In a particular embodiment, the virus-derived assembly protein is a virus-derived GAG protein.

[0021] In particular, the cleavable linker contains three nuclear export signals.

[0022] In a particular embodiment, the fusion protein contains, from N-ter to C-ter, the virus-derived assembly protein, the prime editor and the cleavable linker.

[0023] In a particular embodiment, the protease cleavage sites are selected from Moloney murine leukemia virus (MMLV) protease cleavage sites such as MA / P12, p!2 / CA, CA / NC, NC / PR, RT / IN, or p!2E / p2E, and from Friend murine leukemia virus (FMLV) protease cleavage sites. In a particular embodiment, the napDNAbp is a caspase, in particular is a Cas9, more particularly is a Cas9 nickase.

[0024] In a particular embodiment, the domain comprising a RNA-dependent DNA polymerase activity is a reverse transcriptase, in particular is a MMLV reverse transcriptase or a Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase.

[0025] In a particular embodiment, the reverse transcriptase does not contain a RNaseH domain.

[0026] In a particular embodiment, the prime editor is selected from PE2, PE3, PE4, PE5, PE6, NuPE, TwinPE and PEmax, in particular the prime editor is PE2, PEmax or PE6, such as PE6b, PE6c and PE6d, in particular PE6b.

[0027] In a particular embodiment, the pegRNA comprises two stem-loop structures in 3’, in particular two stem-loop structures selected from tEvoPreQl, CSY4, MS2, TAR, and PP7, Box C / D, Box B or the histone mRNA stem loop, more particularly selected from tEvPreQl, CSY4, MS2, TAR and PP7, more particularly selected from tEvQl, CSY4, TAR and PP7.

[0028] In a particular embodiment of the VLP:

[0029] - the fusion protein contains, from N-ter to C-ter, the virus-derived assembly protein, the prime editor and the cleavable linker,

[0030] - the prime editor is PEmax or PE6, in particular PE6b, PE6c or PE6d, more particularly PE6b, and

[0031] - the pegRNA comprises two stem-loop structures in 3’, in particular selected from tEvQl, CSY4, TAR and PP7.

[0032] In another embodiment of the VLP:

[0033] - the fusion protein contains, from N-ter to C-ter, the virus-derived assembly protein, the prime editor and the cleavable linker,

[0034] - the prime editor is PE6, in particular PE6b, PE6c or PE6d, more particularly PE6b, and

[0035] - the pegRNA comprises two stem-loop structures in 3’, in particular CSY4 and / or TAR, more particularly CSY4 and TAR. In a particular embodiment, the VLP comprises at least two pegRNAs.

[0036] In a particular embodiment, the VLP further comprises at least one target molecule that binds to pegRNA, in particular that binds to one or more of the stem-loop structures of the pegRNA.

[0037] In a particular embodiment, the target molecule is selected from the group consisting of MCP, PP7CP, TAT, Csy4 (wild type or H29A mutant), L7Ae, LambdaN, SLBP, and Com, in particular from the group consisting of MCP, PP7CP, and TAT, more particularly the target molecule is a PP7CP or is a TAT.

[0038] In a particular embodiment, the VLP further comprises one or more additional protein(s), in particular selected from reporter proteins, from helper proteins, and from DNA and / or RNA binding proteins.

[0039] In particular, the additional protein is selected from fluorescent proteins such as mCherry, GFP, mKeita or luciferase; transcription factors; proteins limiting the expression of a surface cell marker in recipient cells; proteins boosting expression of a cell surface marker in recipient cells; proteins generating a selectable phenotype such as proteins inducing a resistance to a drug; recombinases such as CRE; integrases such as BxBl; internal proteins facilitating the incorporation of specific fusogens or viral envelopes; helper proteins including hMLHldn, P53dd, P65, Rad51, T5 exonuclease and FEN1 or binding proteins including MS2 coat protein, PP7 Coat protein, and TATI from HIV.

[0040] In a particular embodiment, the VLP is a retrovirus-derived particle, in particular is a gamma- retrovirus-derived particle. In particular, the VLP is selected in a group comprising Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus-derived vector particles, Caprine arthritis encephalitis virus-derived vector particle, Baboon endogenous virus-derived vector particles, Rabies virus-derived vector particles, Influenza virus-derived vector particles, Norovirus-derived vector particles, Respiratory syncytial virus-derived vector particles, Hepatitis A virus-derived vector particles, Hepatitis B virus-derived vector particles, Hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, Parvovirus-derived vector particles, Papillomavirus-derived vector particles, Yeast retrotransposon-derived vector particles, Measles virus-derived vector particles, and bacteriophage-derived vector particles. In a particular embodiment, the VLP further comprises one or more viral structural protein(s) or viral envelope protein(s), in particular comprising a VSV-G protein, a BAEV envelope or the R- less version of BAEV envelope (BRL), and human syncytin 1 (h-synl), in particularcomprising a VSV-G protein, the R-less version of BAEV envelope (BRL) and (h-synl).

[0041] According to another object, the present invention relates to a plurality of polynucleotides comprising:

[0042] (i) one or more polynucleotide(s) encoding a fusion protein comprising: a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity;

[0043] (ii) a polynucleotide encoding a prime editing guide RNA (pegRNA), in particular a pegRNA bound to the napDNAbp, and

[0044] (iii) optionally a polynucleotide encoding one or more pseudotyping viral envelope protein(s).

[0045] In a particular embodiment, the pegRNA is contained in an intron under the control of a Pol II promoter, in particular of a Pol II CMV promoter.

[0046] In a particular embodiment, the pegRNA is flanked on either side by a nuclease cleaving site, in particular by a csy4 nuclease cleaving site.

[0047] According to another object, the invention relates to a cell line for producing a virus-derived particle comprising the plurality of polynucleotides as described herein.

[0048] According to another object, the invention relates to a composition for altering a target nucleic acid in a eukaryotic cell, which composition comprises virus-derived particle(s) as described herein or a plurality of polynucleotides as described herein.

[0049] The invention further relates to a method for altering a target nucleic acid comprising at least a target sequence in an eukaryotic cell, comprising the steps of: a) bringing into contact the said eukaryotic cell with a virus-derived particle as described herein, or with a plurality of polynucleotides as described herein or with a composition as described herein, and b) collecting the said eukaryotic cell having an altered target nucleic acid.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Fi ure 1 illustrates the results of the PCR assay detecting CTT insertion in the HEK3 locus. From left to right, the PCR wells correspond to HIV-based VLPs (first two wells), VSVG-based VLPs (also called gesicles) (third well), MLV-based VLPs (fourth well) and no VLPs (fifth well).

[0052] Figure 2 illustrates the investigation of the optimal PBS for the YFPs pegRNA. The graph represents the percentage of YFPstop correction (y axis) according to the length of the PBS in nucleotides (x axis). The PBS lengths investigated are, from left to right, 9 nucleotides (nt), l int, 13 nt, 17nt, 24nt and no PBS (control).

[0053] Figure 3 illustrates the percentage of YFP conversion measured by FACS 48h after transduction. The y axis represents the % of YFP conversion and the x axis represents the different VLPs at low cell density (200 000 cells in a 12-w dish) (left columns) and at high density (800000 cells in a 12w-dish) (right columns). From left to right, the VLPs on the x axis represent: VSVG-BRL VLPs, VSVG-BRL syncitin VLPs, VSVG-BRL Spike VLPs, VSVG-BRL HANA M2 VLPs, and no VLPs (control).

[0054] Fi ure 4 illustrates the percentage of YFPstop correction for each of the GAG-PE constructs. The y axis represents the % of YFP conversion and the x axis represents the different GAG-PE constructs. From left to right, the GAG-PE on the x axis represent: GAG-PE VI (VI), GAG-PE V2 (V2), GAG-PE V3 (V3) and GAG-PE V4 (V4). For each construct, three batches were measured: batch #1 (left column), batch #2 (middle column), batch #3 (right column).

[0055] Figure 5 illustrates the % of YFP cells measured in transduced cells after treatment with VLPs loaded with different pegRNA. PegRNAs are from left to right: MS2 Trimmed EvoPreQl, PP7 trimmed evoPreQl, TAR trimmed evoPreQl, the classical epegRNA Trimmed evoPreQl, the pegRNA (without any 3' structure) and the control without PE. Figure 6 illustrates the % of YFP cells measured after transduction for different GAG-PE and pegRNA combinations. Figure 6 A represents the transduction efficiencies measured in reporter cells with VLPs loaded with different evolutions of GAG PE and epegRNAs. Three independent batches were prepared for each condition. Control productions were performed without GAG-PE and non-traduced condition (NTD). The y axis represents the % of YFP conversion and the x axis represents the different GAG-PE and pegRNA combinations. From left to right, the combinations are GAG-PE VI and pegRNA, GAG-PE VI and epegRNA1.2, GAG-PE V3 and epegRNA1.2 and GAG-PE V4 and epegRNA1.2. Figure 6B represents the transduction in different cell lines for each GAG-PE and pegRNA combinations. From left to right, the GAG-PE and pegRNA combinations are GAG-PE VI with pegRNA, GAG-PE V3 with epegRNA1.2, GAG-PE V4 with epegRNA1.2, and GAG-PE V4 with epegRNA1.2. For each combination, the columns represent, from left to right, transduction in HEK293T cells (left column), U2OS cells (middle column) and A549 (right column). For GAG PEV4 with epegRNA1.2, the assay was performed with addition of GAG-MCP (left) or without MCP addition (right).

[0056] Figure 7 illustrates the quantification of pegRNAs relative to epegRNA within YFPs-VLPs (V4) produced with or without addition of capture proteins. The y axis represents the quantification relative to pegRNA and the x axis represents, from left to right, epeg-RNA-MS2 with MCP, ePeg- RNA-MS2 without MCP, ePegRNA-PP7 with PP7PC, ePegRNA-PP7 without PP7PC, ePegRNA-TAR with TATI, ePegRNA-TAR without TATI, ePegRNA-TAR with TAT1A2-26, ePeg alone, PegRNA alone and ePegRNA-MS2-PE. Fold change relative to non-captured epegRNA are indicated.

[0057] Figure 8 illustrates the CTT insertion measured by TIDE in HAP-1 and K562 cells after transduction with PE- VLPs editing HEK3. The y axis represents the % of editing and the x axis represents, from left to right, the HAP1 cells and the K562 cells. For each cell line, the left column represents the % of editing and the right column represents the % of unwanted edits.

[0058] Figure 9 illustrates multiplexing of pegRNA within PE- VLPs. The y axis represents the % of mutated sequence measured by TIDE analysis and the x axis represents the VLP volume (in pL) for each of the HEK locus (CTT) and the RNF2 locus (RNF2), from left to right, lOpL, 20pL and 50pL.

[0059] Figure 10 illustrates the FACS analysis of cells edited by transfection or by VLPs. FACS analysis of cells edited by transfection (bar on the left) or two preparations of PE- VLPs (middle bar and bar on the right). Positions of mismatches are depicted. Results are given as the percentages of editing in recipient cells in a perfect match situation (maximum 100%) (figure 10A), with one mismatch (10%) (figure 10B) or with two mismatches (1%) (figure 10C).

[0060] Figure 11 illustrates the relative CTT insertion in HEK3 locus achieved in HEK293T cells by plasmid transfection or VLP transduction. The y axis represents the relative CTT insertion and the x axis represents, from left to right, different mutated positions in the crRNA and PBS regions: T17, T14, T10, G6, T3, pAl; pA5 and pG9.

[0061] Figure 12 illustrates the structures of the different VLP-PE constructs containing a linker. The bean-shaped form represents the cas9n and the CAS9n is noted with an “M”, the u-shaped form that is either full or has a part of the right side cut-off represents the RT and the RT deltaRNase H respectively (A), the circle represents GAGmlv. The line between the bean-shape and the circle represents a SP linker (no *) or a SP-3NES-SP linker (marked with a *). The curly line connected to certain bean-shapes represents a SP-3NES linker

[0062] Figure 13 illustrates the correction of YFP in cells according to the different VLP constructs. The y axis represents the % of YFP corrected cells and the x axis represents each of the constructs. Three batches were measured for each construct, each represented by a column (dark grey, medium grey and light grey). From left to right, the following constructs are presented: GAG- SP- 3NES-SP CAS9n -RT, GAG- SP-3NES-SP-CAS9n-RTA, GAG- SP- CAS9n -RTmax-SP-3NES, GAG-SP-CAS9n-RTAmax-SP-3NES, GAG- SP-3NES-SP- CAS9n -RTmax, GAG- SP-3NES- SP- CAS9n -RTAmax, GAG- SP- 3NES-SP-CAS9n -RTmax-SP-3NES, GAG- SP- 3NES-SP- CAS9n -RTAmax-SP-3NES, control (untransduced condition).

[0063] Figure 14 illustrates the different steps that are undergone to generate an YFPs epegRNA within the intronic sequence of a luciferase-coding plasmid.

[0064] Figure 15 illustrates the YFP positive cells for each of the tested pegRNAs. The y axis represents the percentage of YFP-positive cells, and the x axis represents, from left to right, the results for the ePegRNAV2, the pegRNA-Csy4, the PegRNA-TAR-Csy4 and the ePegRNAV2-Csy. Three batches were measured for each PegRNA, each represented by a column (dark grey, medium grey and light grey)

[0065] Figure 16 illustrates the YFP corrected cells for V4 and V1.2mp constructs. The y axis represents the % of YFP corrected cells and the x axis represents the V4 construct (left) and the V1.2mp construct (right). Three batches were measured for each construct, each represented by a column (dark grey, medium grey and light grey). Figure 17 illustrates the triplet insertion measured by TIDE for each of the constructs in the HEK3 locus (top figure) or in the RNF2 locus (bottom figure). The y axis represents the % of 3- nucleotide insertion and the x axis represents, from left to right, the following constructs: V4, V4C, V1.2mp and VlmpC.

[0066] Figure 18 illustrates the editing capacity of various versions of PE-VLPs with PE6 editors within the Human HEK3 locus.

[0067] Figure 18 A shows the design of PE-VLP constructs. The constructs were created by fusing the GAG protein from Murine Leukemia Virus (MLV) with PEmax (construct GPmP3n) or various PE6 editors. The constructs are as follows, from the top to the bottom: GPE6b, G3nPPE6b, GPE6bP3n, G3nPPE6bP3n, GPE6c, G3nPPE6c, GPE6cP3n, G3nPPE6cP3n, GPE6d, G3nPPE6d, GPE6dP3n and G3nPPE6dP3n.

[0068] Figure 18 B shows the percentage of insertions of the CTT triplet within the HEK3 locus in HEK293T target cells transduced by a PEmax- VLP and PE6b-VLPs as described in figure 18 A. The y axis represents the % editing. The dark grey bars show the % of 3 -nucleotide insertion (expected) and the light grey bars show the % of indels (unexpected). The x axis represents, from left to right, the following constructs: GPE6b, G3nPPE6b, GPE6bP3n, G3nPPE6bP3n and GPmP3n.

[0069] Figure 18 C shows the percentage of insertion of the Flag sequence (24 nucleotides) within the HEK3 locus in HEK293T cells transduced by a PEmax- VLP and PE6b-VLPs as described in figure 18 A. The y axis represents the % editing. The dark grey bars show the % of 3 -nucleotide insertion (expected) and the light grey bars show the % of indels (unexpected). The x axis represents, from left to right, the following constructs: GPE6b, G3nPPE6b, GPE6bP3n, G3nPPE6bP3n and GPmP3n.

[0070] Figure 18 D compares the editing efficiency of a PEmax- VLP with PE6c-VLPs and PE6d-VLPs as described in figure 18 A for the insertion of a pinned 25 nucleotide sequence within the human HEK3 locus. The y axis represents the % editing. The dark grey bars show the % of 25-nucleotide insertion (expected) and the light grey bars show the % of indels (unexpected). The x axis represents, from left to right, the following constructs: GPE6c, G3nPPE6c, GPE6cP3n, G3nPPE6cP3n, GPE6d, G3nPPE6d, GPE6dP3n, G3nPPE6dP3n and GPmP3n.

[0071] Figure 18 E compares the editing efficiency of PEmax- VLPs with PE6c-VLPs and PE6d-VLPs as described in figure 18 A for the insertion of an unpinned 25 nucleotide sequence within the human HEK3 locus. The y axis represents the % editing. The dark grey bars show the % of 25-nucleotide insertion (expected) and the light grey bars show the % of indels (unexpected). The x axis represents, from left to right, the following constructs: GPE6c, G3nPPE6c, GPE6cP3n, G3nPPE6cP3n, GPE6d, G3nPPE6d, GPE6dP3n, G3nPPE6dP3n and GPmP3n.

[0072] DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention relates to a virus-derived particle that is able to deliver the Prime editing tools necessary for genetic engineering. The invention further provides a plurality of polynucleotides encoding said Prime editing tools, a cell line for producing a VLP, a composition for altering a target nucleic acid in a eukaryotic cell as well as a method for altering a target nucleic acid comprising at least a target sequence in a eukaryotic cell.

[0074] Surprisingly, the inventors have shown that they were able to produce VLPs able to efficiently deliver the necessary Prime Editing tools in target cells. In particular, the inventors were able to provide VLPs wherein the PE tools were adapted to increase the efficiency of delivery compared to the PE tools known in the art.

[0075] As such, the present inventors have conceived a powerful method to transfer the Prime editing active machinery within human and other mammalian cells, including induced pluripotent stem cells (IPSCs) and stem cells, by using versatile virus-derived particles (which are also termed “Virus Like Particles” or “VLPs” herein).

[0076] The inventors have further shown that optimized versions of the VLPs of the invention ensure a transient delivery of the PE machinery into different target cells and boosted by close to 15-fold the editing efficiencies of the system as compared with the original PE system.

[0077] Further, it has been demonstrated that PE- VLPs of the invention can also codeliver, into different human cell-lines, including human iPSc, different pegRNAs alone or in combination and can host other proteins of interest.

[0078] Overall, the inventors were able to demonstrate that the molecular improvements of constructs of the Prime editing tool enabled enhanced fidelity of editing, in particular compared to the existing tools.

[0079] To the extent of their knowledge, such improvements to the Prime Editing tools have never been implemented in the art, in particular in the specific context of VLP delivery systems.

[0080] Virus-derived particles

[0081] As used herein, a virus-derived particle consists of a virus-like particle formed by one or more virus-derived protein(s), which virus-derived particle is substantially devoid of any nucleic acid encoding a nucleic acid or a protein of interest, or alternatively is devoid of any nucleic acid encoding a nucleic acid or a protein of interest. Notably, a virus-derived particle according to the invention is substantially devoid of any nucleic acid encoding a viral nucleic acid or a viral protein of interest, or alternatively is devoid of any nucleic acid encoding a viral nucleic acid or a viral protein of interest. A virus-derived particle according to the invention is replicationincompetent.

[0082] As used herein “VLP production” refers to the packaging of useful material (proteins / RNP) in producer cells. It is an assembly step.

[0083] “VLP-delivery” refers classically to the process that implies a VLP entering into the cells and the release of the VLP’s cargo and its translocation to its subcellular relevant location.

[0084] “PE delivery” refers to the integrated effects of both processes, VLP production followed by VLP delivery.

[0085] Virus-derived particles

[0086] Any virus suitable for gene therapy may be used, including but not limited to adeno-associated virus (“AAV”); adenovirus; herpes virus; lentivirus and retrovirus. Adeno-associated virus (“AAV”) may be selected in a group comprising AAV1, AAV6, AAV7, AAV8, AAV9 or rhlO, which AAV are particularly suitable for use in human subjects.

[0087] The general methods that are known in the art for producing viral vector particles, which generally contain coding nucleic acids of interest, may also be used for producing the virus- derived particles according to the present invention, which do not contain coding nucleic acids of interest.

[0088] Conventional viral vector particles encompass retroviral, lentiviral, adenoviral and adeno- associated viral vector particles that are well known in the art. For a review of various viral vector particles that may be used, the one skilled in the art may notably refer to Kushnir et al. (2012, Vaccine, Vol. 31: 58-83), Zeltons (2013, Mol Biotechnol, Vol. 53: 92-107), Ludwig et al. (2007, Curr Opin Biotechnol, Vol. 18(n°6): 537-55) and Naskalaska et al. (2015, Vol. 64 (n°l): 3-13). Further, references to various methods using virus-derived particles for delivering proteins to cells are found by the one skilled in the art in the article of Maetzig et al. (2012, Current Gene therapy, Vol. 12: 389-409) as well as the article of Kaczmarczyk et al. (2011, Proc Natl Acad Sci USA, Vol. 108 (n° 41): 16998-17003).

[0089] Generally, a virus-derived particle that is used according to the invention, which virus-derived particle may also be termed “Virus-Like Particle” or “VLP”, is formed by one or more virus- derived structural protein(s) and / or one more virus-derived envelope protein. Throughout this text, “Virus-Like Particle” and “Virus-Derived Particle” are used interchangeably.

[0090] A virus-derived particle that is used according to the present invention is replication incompetent in a host cell wherein it has entered.

[0091] In preferred embodiments, a virus-derived particle is formed by one or more retrovirus-derived structural protein(s) and optionally one or more virus-derived envelope protein(s).

[0092] In preferred embodiments, the virus-derived structural protein is a retroviral gag protein or a peptide fragment thereof. As it is known in the art, Gag and Gag / pol precursors are expressed from full length genomic RNA as polyproteins, which require proteolytic cleavage, mediated by the retroviral protease (PR), to acquire a functional conformation. Further, Gag, which is structurally conserved among the retroviruses, is composed of at least three protein units: matrix protein (MA), capsid protein (CA) and nucleocapsid protein (NC), whereas Pol consists of the retroviral protease, (PR), the retrotranscriptase (RT) and the integrase (IN).

[0093] In some embodiments, a virus-derived particle comprises a retroviral Gag protein but does not comprise a Pol protein.

[0094] As it is known in the art, the host range of retroviral vector, including lentiviral vectors, may be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of viral vector particles bearing glycoproteins derived from other enveloped viruses. Such pseudotyped viral vector particles possess the tropism of the virus from which the glycoprotein is derived.

[0095] In some embodiments, a virus-derived particle is a pseudotyped virus-derived particle comprising one or more viral structural protein(s) or viral envelope protein(s) imparting a tropism to the said virus-derived particle for certain eukaryotic cells. A pseudotyped virus-derived particle as described herein may comprise, as the viral protein used for pseudotyping, a viral envelope protein selected in a group comprising VSV-G protein, Measles virus HA protein, Measles virus F protein, Influenza virus HANA protein, Moloney virus MLV-A protein, Moloney virus MLV-E protein, Baboon Endogenous retrovirus (BAEV) envelope protein, HIV envelope proteins (as documented for VLP by Hamilton et al Cell report 2021), Ebola virus glycoprotein and foamy virus envelope protein, Sendai virus envelope proteins or a combination of two or more of these viral envelope proteins.

[0096] This may also include natural fusogens including human or simian Syncitins, myogenic cell fusogens (Hindi et al 2023), fusogens of sperm cell (Izumo), tetraspanin CD9 (as documented by Bblker et al Mol Ther 2018), programmable envelope proteins that can be directed toward specific targets with antibodies (Strebinger et al Nat Comm 2023, Hamilton et al. Nat Biotech 2024), with Nanobodies or with drugs.

[0097] A well-known illustration of pseudotyping viral vector particles consists of the pseudotyping of viral vector particles with the vesicular stomatitis virus glycoprotein (VSV-G). For the pseudotyping of viral vector particles, the one skilled in the art may notably refer to Yee et al. (1994, ProcNatl Acad Sci, USA, Vol. 91: 9564-9568) Cronin et al. (2005, Curr Gene Ther, Vol. 5(n°4): 387-398).

[0098] In a particular embodiment, the VLP comprises VSV-G protein, a BAEV envelope or the R-less version of BAEV envelope (BRL), and human syncytin-1 (h-synl). In another embodiment, the VLP comprises VSV-G protein, the R-less version of BAEV envelope (BRL) and h-synl.

[0099] BAEV envelope and the R-less version of BAEV envelope (BRL) are described for efficient gene transfer for example in Girard-Gagnepain A et al. (2014. Blood. 2014 Aug 21;124(8): 1221-31. doi: 10.1182 / blood-2014-02-558163. Epub 2014 Jun 20).

[0100] For producing virus-derived particles, and more precisely VSV-G pseudotypes virus-derived particles, for delivering protein(s) of interest into target cells, the one skilled in the art may refer to Mangeot et al. (2011, Molecular Therapy, Vol. 19 (n°9): 1656-1666).

[0101] In some preferred embodiments, the VSV-G protein which is used for pseudotyping a virus- derived particle of the invention has the amino acid sequence of SEQ ID NO: 1, that may be encoded by a nucleic acid comprising the sequence of SEQ ID NO: 2. In some preferred embodiments, the BAEV-G (BAEV) protein which is used for pseudotyping a virus-derived particle of the invention has the amino acid sequence of SEQ ID NO: 3, that may be encoded by a nucleic acid comprising the sequence of SEQ ID NO: 4.

[0102] In some preferred embodiments, a human syncitin protein is included in the VLP. In particular, the human syncitin protein which is used for pseudotyping a virus-derived particle of the invention has the amino acid sequence of SEQ ID NO: 5, that may be encoded by a nucleic acid having the sequence of SEQ ID NO: 6.

[0103] Thus, in some embodiments, a virus-derived particle further comprises a viral envelope protein, wherein either (i) the said viral envelope protein originates from the same virus as the viral structural protein, e.g. originates from the same virus as the viral Gag protein, or (ii) the said viral envelope protein originates from a virus distinct from the virus from which originates the viral structural protein, e.g. originates from a virus distinct from the virus from which originates the viral Gag protein.

[0104] As it is readily understood by the one skilled in the art, a virus-derived particle that is used according to the invention may be selected in a group comprising Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus- derived vector particles, Caprine arthritis encephalitis virus-derived vector particle, Baboon endogenous virus-derived vector particles, Rabies virus-derived vector particles, Influenza virus- derived vector particles, Norovirus-derived vector particles, Respiratory syncytial virus-derived vector particles, Hepatitis A virus-derived vector particles, Hepatitis B virus-derived vector particles, Hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, Parvovirus-derived vector particles, Papillomavirus-derived vector particles, Yeast retrotransposon-derived vector particles, Measles virus-derived vector particles, and bacteriophage-derived vector particles.

[0105] In particular, a virus-derived particle that is used according to the invention is a retrovirus-derived particle. Such retrovirus may be selected among Moloney murine leukemia virus, Bovine immunodeficiency virus, Simian immunodeficiency virus, Feline immunodeficiency virus, Human immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis encephalitis virus. In another embodiment, a virus-derived particle that is used according to the invention is a gamma-retrovirus-derived particle. Lentiviruses belong to the retroviruses family, and have the unique ability of being able to infect non-dividing cells.

[0106] Such lentivirus may be selected among Bovine immunodeficiency virus, Simian immunodeficiency virus, Feline immunodeficiency virus, Human immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis encephalitis virus.

[0107] For preparing Moloney murine leukemia virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Sharma et al. (1997, Proc Natl Acad Sci USA, Vol. 94: 10803+-10808), Guibingua et al. (2002, Molecular Therapy, Vol. 5(n°5): 538-546). Moloney murine leukemia virus-derived (MLV-derived) vector particles may be selected in a group comprising MLV-A-derived vector particles and MLV-E-derived vector particles.

[0108] For preparing Bovine Immunodeficiency virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Rasmussen et al. (1990, Virology, Vol. 178(n°2): 435-451)

[0109] For preparing Simian immunodeficiency virus-derived vector particles, including VSV-G pseudotyped SIV virus-derived particles, the one skilled in the art may notably refer to the methods disclosed by Mangeot et al. (2000, Journal of Virology, Vol. 71(n°18): 8307-8315), Negre et al. (2000, Gene Therapy, Vol. 7: 1613-1623) Mangeot et al. (2004, Nucleic Acids Research, Vol. 32 (n° 12), el02)

[0110] For preparing Feline Immunodeficiency virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Saenz et al. (2012, Cold Spring Harb Protoc, (1): 71-76; 2012, Cold Spring Harb Protoc, (1): 124-125; 2012, Cold Spring Harb Protoc, (1): 118- 123).

[0111] For preparing Human immunodeficiency virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Jalaguier et al. (2011, PlosOne, Vol. 6(n°l l), e28314), Cervera et al. (J Biotechnol, Vol. 166(n°4): 152-165), Tang et al. (2012, Journal of Virology, Vol. 86(n°14): 7662-7676)

[0112] For preparing Equine infection anemia virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Olsen (1998, Gene Ther, Vol. 5(n°l 1): 1481-1487). For preparing Caprine arthritis encephalitis virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Mselli-Lakhal ety al. (2006, J Virol Methods, Vol. 136(n°l-2): 177-184).

[0113] For preparing Baboon endogenous virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Girard-Gagnepain et al. (2014, Blood, Vol. 124(n°8): 1221-1231)

[0114] For preparing Rabies virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Kang et al. (2015, Viruses, Vol. 7: 1134-1152, doi: 10.3390 / v7031134), Fontana et al. (2014, Vaccine, Vol. 32(n°24): 2799-27804) or to the PCT application published under n° WO 2012 / 0618.

[0115] For preparing Influenza virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Quan et al. (2012, Virology, Vol. 430: 127-135) and to Latham et al. (2001, Journal of Virology, Vol. 75(n°13),: 6154-6155).

[0116] For preparing Norovirus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Tome-Amat et al., (2014, Microbial Cell Factories, Vol. 13: 134-142).

[0117] For preparing Respiratory syncytial virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Walpita et al. (2015, PlosOne, D01:10.1371 / joumal.pone.0130755)

[0118] For preparing Hepatitis B virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Hong et al. (2013, Vol. 87(n°12): 6615-6624).

[0119] For preparing Hepatitis E virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Li et al. (1997, Journal of Virology, Vol. 71(n°10): 7207-7213).

[0120] For preparing Newcastle disease virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Murawski et al. (2010, Journal of Virology, Vol. 84(n°2): 1110-1123)

[0121] For preparing Norwalk virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Herbst-Kralovetz et al. (2010, Expert Rev Vaccines, Vol. 9(n°3): 299-307). For preparing Parvovirus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Ogasawara et al. (2006, In Vivo, Vol. 20: 319-324)

[0122] For preparing Papillomavirus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Wang et al. (2013, Expert Rev Vaccines, Vol. 12(n°2): doi:10.1586 / erv,12.151)

[0123] For preparing Yeast retrotransposon-derived vector particles, the one skilled in the art may refer to the methods disclosed by Peifang et al. (1994, Clin Exp Immunol, Vol. 97(n°3): 361-366) or to the US patent n° US 6,060,064

[0124] For preparing Measles virus-derived vector particles, the one skilled in the art may notably refer to the methods disclosed by Brandler et al. (2008, Vol. 31(n°2-3): 271-291).

[0125] For preparing bacteriophage-derived vector particles, and in particular Q-beta virus-like particles, the one skilled in the art may notably refer to the methods disclosed by Brown et al. (2009, Biochemistry, Vol. 48(n°47): 11155-11157).

[0126] A virus-derived particle that is used herein comprises a Gag protein, and most preferably a Gag protein originating from a virus selected in a group comprising Rous Sarcoma Virus (RSV) Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Moloney Murine Leukemia Virus (MLV) and Human Immunodeficiency Viruses (HIV-1 and HIV-2) especially Human Immunodeficiency Virus of type 1 (HIV-1).

[0127] In some embodiments, a virus-derived particle may also comprise one or more viral envelope protein(s). The presence of one or more viral envelope protein(s) may impart to the said virus- derived particle a more specific tropism for the cells which are targeted, as it is known in the art. The one or more viral envelope protein(s) may be selected in a group comprising envelope proteins from retroviruses, envelope proteins from non-retroviral viruses, and chimeras of these viral envelope proteins with other peptides or proteins. An example of a non-lentiviral envelope glycoprotein of interest is the lymphocytic choriomeningitis virus (LCMV) strain WE54 envelope glycoprotein. These envelope glycoproteins increase the range of cells that can be transduced with retroviral derived vectors.

[0128] In some preferred embodiments, the virus-derived particle comprises a Gag protein originating from a virus selected in a group comprising Rous Sarcoma Virus (RSV) and Moloney Murine Leukemia Virus (MLV). In some preferred embodiments, a virus-derived particle that is used herein, further comprises a pseudotyping viral envelope protein, and most preferably a VSV-G protein.

[0129] Fusion protein

[0130] Virus-derived assembly protein

[0131] A virus-derived particle according to the invention comprises a fusion protein. Said fusion protein comprises at least one virus-derived assembly protein.

[0132] Virus-derived assembly proteins are proteins involved in virus assembly. This phenomenon is a dynamic process driven by genetically programmed sequential morphogenetic reactions involving protein-protein associations and interactions between the viral genome and capsid proteins.

[0133] Virus-derived assembly proteins are described further above. In particular, virus-derived assembly proteins may be selected from viral structural protein(s) or viral envelope protein(s), in particular as described above. Virus-derived assembly proteins may include proteins responsible for the assembly of human viruses and also proteins expressed in humans that can autoassemble to form VLP particles including human PEG10 (as documented by SEGEL et al.) and GAG proteins from Human Endogenous Virus (HERV).

[0134] In a particular embodiment, the virus-derived assembly protein is a virus-derived GAG protein.

[0135] In a particular embodiment, the virus-derived assembly protein is a virus-derived fragment of the GAG protein. GAG is a composite protein with Matrix-Capsid-Nucleocapsid (MACANC). A fragment of the GAG protein includes the MA-CA fragment, the MA fragment and the MA-NC fragment. Fragments of the GAG proteins have been successfully used in Voelkel et al. (2010, PNAS, 107 (17) 7805-78).

[0136] In some embodiments, the said Gag protein is encoded by an expression cassette selected in a group comprising an expression cassette encoding a GAG-PRO-POL polyprotein and an expression cassette encoding a GAG protein. In another embodiment, the said Gag protein is encoded by an expression cassette selected in a group comprising an expression cassette encoding a GAG-PRO or GAG-PRO-RT polyproteins and an expression cassette encoding a GAG protein.

[0137] Indeed, in some embodiments, virus-derived particles are formed in packaging cells expressing a Gag-Pro-Pol viral protein. Without wishing to be bound by any particular theory, the inventors believe that in these embodiments, the Pro protein (i.e. the viral protease) is active in the virus- derived particles and cleaves the fusion protein, typically the Gag-PE fusion protein, so as to generate the free Cas protein, especially the free PE.

[0138] In a particular embodiment, GAG-PRO-POL may be encoded by a plasmid derived from FMLV which ensure the assembly of MLV-derived VLPs that can incorporate a fusion protein GAGmlv- X where X can be a protein of interest, in particular can be PE. The MLV protease cleaves a GAG-PRO-POL sequence and a PS specific to MLV. Such a cassette may have a NCBI Reference Sequence: NP 040332.1. The same may apply for GAG-PRO and for GAG-PRO-RT.

[0139] According to another embodiment, GAG-PRO-POL from HIV can be used and will assist the production of HIV-derived VLPs that can incorporate a fusion protein GAGhiv-X, wherein X is as described above. The same may apply for GAG-PRO and for GAG-PRO-RT.

[0140] According to another embodiment, GAG-PRO-POL from SIV can be used and will assist the production of SIV-derived VLPs that can incorporate a fusion protein GAGsiv-X, wherein X is as described above. The same may apply for GAG-PRO and for GAG-PRO-RT.

[0141] Cleavable linker

[0142] A virus-derived particle according to the invention comprises a fusion protein comprising a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical.

[0143] A nuclear export signal (NES) is a short target peptide containing 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. The NES is recognized and bound by exportins. Examples of NES include classic NES (also known as the “canonical” NES), PKI NES, HIV-1 Tev NES and MAPKK NES.

[0144] In a particular embodiment, the cleavable linker contains one NES, in particular contains two NES, more particularly contains three NES. In a particular embodiment, the cleavable linker does not contain more than three NES.

[0145] In a particular embodiment, a NES included in the cleavable linker has the following sequence: SEQ ID NO: 7 (LPPLERLTL) In a particular embodiment, the three NES included in the cleavable linker have the following sequence: SEQ ID NO: 8 (LPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTL).

[0146] The cleavable linker of the invention further comprises a first protease cleavage site and optionally a second protease cleavage site.

[0147] Protease cleavage sites are amino acid sequences recognized by specific proteases as an enzymatic hydrolysis site.

[0148] Protease cleavage sites, or proteolytic sites, which may also be termed protease sites, are well known from the one skilled in the art. A protease cleavage site that may be contained in the cleavable linker may be a site that is cleavable by a protease selected in a group comprising trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), endoproteinase Glu C (EC 3.4.21.19), endoproteinase Lys-C (EC 3.4.21.50), pepsin (EC 3.4.23.1), elastase (EC 3.4.21.36) abd carboxypeptidase (EC 3.4.17.1).

[0149] In a particular embodiment, the protease cleavage sites are selected from Moloney murine leukemia virus (MMLV) protease cleavage sites such as MA / P12, pl2 / CA, CA / NC, NC / PR, RT / IN, or pl2E / p2E or from Friend murine leukemia virus (FMLV) protease cleavage sites.

[0150] MMLV protease cleavage sites are described in Feher et al. J Gen Virol 2006.

[0151] In a particular embodiment, the protease cleavage site is the MA / P12 site of MLV and has the following sequence: PRSSLYPALTP (SEQ ID NO: 9).

[0152] According to a particular embodiment, the fusion protein contains, from N-ter to C-ter, the virus- derived assembly protein, the cleavable linker and the prime editor. In other words, the cleavable linker is localized between the virus-assembly protein and the prime editor.

[0153] In particular, when the cleavable linker is localized between the virus-assembly protein and the prime editor, the cleavable linker contains, from N-ter to C-ter, a first protease cleavage site, at least one NES, in particular three NES, and a second protease cleavage site.

[0154] The sequence of the cleavable linker according to this embodiment is, for example: TRSSLYPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTLARPQTRSSLYPAL TP (SEQ ID NO: 10). According to another embodiment, the fusion protein contains, from N-ter to C-ter, the virus- derived assembly protein, the prime editor and the cleavable linker. In other words, the cleavable linker is localized in C-ter of the fusion protein.

[0155] In particular, when the cleavable linker is localized in C-ter of the fusion protein, the cleavable linker contains, from N-ter to C-ter, a single protease cleavage site and at least one NES, in particular three NES.

[0156] In this embodiment, the fusion protein further contains, between the virus-derived assembly protein and the prime editor, at least one protease cleavage site. Suitable protease cleavage sites are well known to those skilled in the art and described elsewhere in the text.

[0157] The sequence of the cleavable linker according to this embodiment is, for example: TRSSLYPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTL (SEQ ID NO: 11).

[0158] In another embodiment, the fusion protein contains, from N-ter to C-ter, the virus-derived assembly protein, a first cleavable linker, the prime editor and a second cleavable linker. In this embodiment, the first cleavable linker contains, from N-ter to C-ter, a first protease cleavage site, at least one NES, in particular three NES, and a second protease cleavage site. The second cleavable linker contains, from N-ter to C-ter, a single protease cleavage site and at least one NES, in particular three NES.

[0159] In a particular embodiment, the first and second cleavable linkers are identical.

[0160] In a particular embodiment, the first and second cleavable linkers are different. In this particular embodiment, the first cleavable linker may have an amino acid sequence comprising, in particular consisting of, the sequence set forth as SEQ ID NO: 10 and the second cleavable linker may have an amino acid sequence comprising, in particular consisting of, the sequence set forth as SEQ ID NO: 11.

[0161] In an alternative embodiment, the fusion protein does not contain a cleavable linker as described above. In this embodiment, the fusion protein may comprise, from N-ter to C-ter, the virus- derived assembly protein, at least one protease cleavage site (PS) and the prime editor.

[0162] Protease cleavage sites that may be used according to this embodiment are described elsewhere in the specification. Prime Editor

[0163] A virus-derived particle according to the invention comprises a fusion protein comprising a Prime Editor. A Prime Editor (or PE) according to the invention comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity. The term “prime editor” refers to fusion constructs comprising a napDNAbp and a reverse transcriptase and is capable of carrying out prime editing on a target nucleotide sequence in the presence of a PEgRNA (or “extended guide RNA” pr “pegRNA”). The term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a PEgRNA, and / or further complexed with a second-strand nicking sgRNA. In some embodiments, the prime editor may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a PEgRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein.

[0164] The term “primer binding site” or “the PBS” refers to the nucleotide sequence located on a PEgRNA as a component of the extension arm (typically at the 3 ’ end of the extension arm) and serves to bind to the primer sequence that is formed after nicking of the target sequence by the prime editor. As detailed elsewhere, when the nickase component of a prime editor nicks one strand of the target DNA sequence, a 3 ‘-ended ssDNA flap is formed, which serves a primer sequence that anneals to the primer binding site on the PEgRNA to prime reverse transcription.

[0165] A target sequence as referred to herein, is a sequence to be edited in a cell, in particular in an eukaryotic cell. The target sequence is localized in a target nucleic acid.

[0166] Prime editing is a novel genetic engineering system that allows targeted rewriting of genome with very limited risk of off-target effects, described in Anzalone et al. (Nature volume 576, pages 149-157 (2019)).

[0167] In a particular embodiment, the napDNAbp is a caspase. In a particular embodiment, the napDNAbp is a Cas9. In a particular embodiment, the Cas9 is a Cas9 nickase.

[0168] In a particular embodiment, the napDNAbp is a Cas9 H840A nickase.

[0169] The Cas9 H840A nickase may have a nucleic acid sequence of SEQ ID NO: 12. The Cas9 H840A nickase may have an amino acid sequence of SEQ ID NO: 13.

[0170] The Cas9 enzyme contains two nuclease domains that can cleave DNA sequences, a RuvC domain that cleaves the non-target strand and a HNH domain that cleaves the target strand. The introduction of a H840A substitution in Cas9, through which the 840thamino acid histidine is replaced by an alanine, inactivates the HNH domain. With only the RuvC functioning domain, the catalytically impaired Cas9 introduces a single strand nick, hence the name nickase.

[0171] In another embodiment, the napDNAbp is a Cas9 comprising one or more mutations selected from the group consisting of: R221K, N394K, K775R, K918A, H99R, E471K, I632V, D645N, H721Y, R654C and H840A.

[0172] In another embodiment, the napDNAbp is a Cas9 nickase comprising at least mutations R221K, N394K and H840A.

[0173] In a preferred embodiment, the napDNAbp is a Cas9 nickase comprising mutations R221K, N394K and H840A.

[0174] In another embodiment, the napDNAbp is a Cas9 nickase comprising mutations K775R, K918A and H840A.

[0175] In another embodiment, the napDNAbp is a Cas9 nickase comprising mutations H840A, H99R, E471K, I632V, D645N, H721Y and K918A.

[0176] In another embodiment, the napDNAbp is a Cas9 nickase comprising mutations H840A, H99R, E471K, I632V, D645N, R654C and H721Y.

[0177] According to a particular embodiment, the domain comprising a RNA-dependent DNA polymerase activity is a reverse transcriptase.

[0178] Reverse transcriptases suitable according to the invention include Moloney Murine Leukemia Virus (MMLV) transcriptase, Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase, HFV reverse transcriptase, LtrA reverse transcriptase, HERV-Koon reverse transcriptase, Tel4c reverse transcriptase, marathon reverse transcriptase, GslIIC, Ma-Int5, Vp96, Er RT, MPMV, SRV2, GALV, Ty3, MMTV, PERV, BAEMV, AVIRE, WMSV, KORV, and reverse transcriptase domains from endogenous elements of the human genome such as LINE or HERV.

[0179] Such reverse transcriptase are described in Doman et al. (Cell. 2023 Aug 31; 186(18): 3983- 4002. e26., doi: 10.1016 / j. cell.2023.07.039) and in Griinewald J, et al. (Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat Biotechnol. 2023 doi: 10.1038 / s41587-022-01473-l). In a particular embodiment, the domain comprising a RNA-dependent DNA polymerase activity is a Moloney Murine Leukemia Virus (MMLV) transcriptase.

[0180] The MMLV transcriptase is an enzyme that synthesizes DNA from a single-stranded RNA template.

[0181] In a particular embodiment, the domain comprising a RNA-dependent DNA polymerase activity is a Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase.

[0182] The Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase generates its own RNA self-primer with its encrypted RNaseH activity. After the self-primer is generated, the Tfl reverse transcriptase extends it using its DNA polymerase activity to synthesize the negativesense DNA strand.

[0183] In particular, the domain comprising an RNA-dependent DNA polymerase activity is a Moloney Murine Leukemia Virus (MMLV) transcriptase or is a Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase.

[0184] In a particular embodiment, the prime editor is a fusion protein containing the nucleic acid programmable DNA binding protein (napDNAbp) and the domain comprising a RNA-dependent DNA polymerase activity.

[0185] In a particular embodiment, the prime editor is a fusion protein consisting of a Cas9 H840A nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase. Such prime editors are also described in Anzalone et al. (2019).

[0186] In a particular embodiment, the prime editor is a fusion protein consisting of a Cas9 nickase fused to a Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase. Such prime editors are also described in Doman et al. (Cell. 2023 Aug 31; 186(18): 3983-4002.e26., doi: 10.1016 / j.cell.2023.07.039).

[0187] In a particular embodiment, the reverse transcriptase does not contain a RNaseH domain. This domain induces degradation of RNA during reverse transcription.

[0188] According to a particular embodiment, the prime editor is a fusion protein consisting of a Cas9 H840A nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase that does not contain a RNaseH domain. Such a prime editor may be called PE2Amh in the present text. Different prime editors are known in the art.

[0189] PEI comprises a wild-type Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase fused to the Cas9 H840A nickase C-terminus.

[0190] PE2 is an evolution of PEI wherein a mutant M-MLV RT was incorporated into PEI to give rise to (Cas9 (H840A)-M-MLV RT(D200N / L603W / T330P / T306K / W313F).

[0191] PE3 is an evolution of PE2 wherein a sgRNA designed to match the edited sequence introduced by the pegRNA, but not the original allele, is introduced. It directs the Cas9 nickase portion of the fusion protein to nick the unedited strand at a nearby site, opposite to the original nick. Nicking the non-edited strand causes the cell’s natural repair system to copy the information in the edited strand to the complementary strand, permanently installing the edit.

[0192] PE4 utilizes the same machinery as PE2, but also includes a plasmid that encodes for dominant negative MMR protein MLH1. Dominant negative MLH1 is able to essentially knock out endogenous MLH1 by inhibition, thereby reducing cellular MMR response and increasing prime editing efficiency.

[0193] PE5 utilizes the same machinery as PE3, but also includes a plasmid that encodes for dominant negative MLH1. Like PE4, this allows for a knockdown of endogenous MMR response, increasing the efficiency of prime editing.

[0194] PE6 consists of evolved editors derived from phage-assisted continuous evolution. They are small and may be more efficient. Such editors are described in Doman et al. (Cell. 2023 Aug 31; 186(18): 3983-4002. e26., doi: 10.1016 / j. cell.2023.07.039). Several variants of the PE6 prime editor exist and have been described in the art. By PE6, it is understood herein any one of the PE6 variants PE6a, PE6b, PE6c, PE6d, PE6e, PE6f or PE6g.

[0195] PE6a is a prime editor variant that utilizes a highly compact reverse transcriptase (RT) domain derived from the evo-Ec48 RT from E. coli. This RT contains 8 substitutions acquired through the PACE and PANCE methods compared to the WT variant (i.e. substitutions E60K, K87E, E165D, D243N, R267I, E279K, K318E and K343N). The RT is truncated and engineered for minimal size (approximately 1.2 kb), retaining essential polymerase. The Cas9 nickase is a SpCas9 comprising mutations R221K, N394K and H840A (i.e. The SpCas9 from PEmax). Its small size makes it especially suitable for delivery via size-limited vectors such as AAV. PE6b incorporates a slightly larger RT domain than PE6a (about 1.5 kb) derived from the Schizosaccharomyces pombe Tfl retrotransposon. This RT has been evolved with the PANCE method and presents numerous amino acid substitutions (i.e. substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E and S492N). The Cas9 component is unchanged from PE6a, but the RT’s origin and structure differ, enabling compatibility with a broader range of pegRNA designs. PE6b may for example be obtained using the Addgene plasmid # 207852 (http: / / n2t.net / addgene:207852).

[0196] PE6c features a full-length Tfl RT fused to SpCas9(H840A), but with additional mutations identified through phage-assisted continuous evolution (PACE) and phage-assisted noncontinuous evolution (PANCE) to enhance processivity and compatibility with structured reverse transcription templates (RTTs). The Cas9 component is unchanged from PE6b. The Tfl RT contains 15 substitutions compared to the WT Tfl RT and 3 of them are only found in this variant (i.e. these substitutions are absent in PE6b). Those additional substitutions are rationally designed (P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, S188K, I260L, F269L, S297Q, A363V, K413E and S492N; in bold are the novel substitutions of PE6c as compared to PE6b). It enables efficient installation of longer and structured (<-23 kcal. mol'1) DNA sequences. PE6c may for example be obtained using the Addgene plasmid # 207853

[0197] (http : / / n2t. net / addgene : 207853 ) .

[0198] PE6d is based on an evolved and engineered MMLV RT (the one from PEmax). The substitutions are listed here: T128N, D200C, V223Y, T306K, W313F, T330P and L603W (in bold are the mutations specific to evoMMLV RT as compared to the MMLV RT of PEmax). Furthermore, the RNaseH domain is deleted (which is not necessarily the case in PEmax or PE2). These mutations were selected for their ability to enable efficient installation of longer and structured DNA sequences. The rest of the construct, including the Cas9 and linker regions, is consistent with PE6c. PE6d may for example be obtained using the Addgene plasmid # 207860 (http : / / n2t. net / addgene : 207860) .

[0199] PE6e, PE6f, and PE6g are variants that introduce targeted mutations into the Cas9 domain, rather than the RT. PE6e has a SpCas9 domain with mutations K775R, K918A and H840A as compared to the SpCas9 domain of PEmax, specifically targeting residues implicated in DNA binding and nicking activity. PE6f follows the same general design as PE6e, but comprises the following mutations in the Cas9 domain: H840A, H99R, E471K, I632V, D645N, H721Y and K918A. PE6g represents a variant incorporating a distinct set of Cas9 mutations: H840A, H99R, E471K, 163 IN, D645N, R654C and H721Y. NuPE (Nuclease Prime Editor) uses Cas9 nuclease instead of Cas9(H840A) nickase. Unlike prime editor 3 (PE3) that requires dual-nick at both DNA strands to induce efficient prime editing, Nuclease Prime Editor requires only a single pegRNA since the single-gRNA already creates double-strand break instead of single-strand nick.

[0200] The “twin prime editing” (twinPE) mechanism reported in 2021 allows editing large sequences of DNA - sequences as large as genes - which addresses the method’s key drawback. It uses a prime editor protein and two prime editing guide RNAs.

[0201] Suitable prime editors are described in Chen, P.J., Liu, D.R. Prime editing for precise and highly versatile genome manipulation. Nat Rev Genet 24, 161-177 (2023).

[0202] In particular, a prime editor suitable according to the invention is selected from the list consisting of PEI, PE2, PE3, PE4, PE5, PE6, NuPE, TwinPE and PEmax, in particular the prime editor is PE2 or PEmax.

[0203] In particular, a prime editor suitable according to the invention is selected from the list consisting of PEI; PE2; PE3; PE4; PE5; PE6, in particular selected in the group consisting of PE6a, PE6b, PE6c, PE6d, PE6e, PE6f and PE6g; NuPE; TwinPE; and PEmax. In particular, the prime editor is PE2; PEmax; or PE6, more particularly PE6b, PE6c or PE6d; even more particularly the prime editor is PE2, PEmax or PE6b.

[0204] In a particular embodiment, the prime editor is PEmax.

[0205] PEmax consists of an optimized prime editor that uses a human codon-optimized RT, a 34-aa linker containing a bipartite SV40 NLS, an additional C-terminal c-Myc NLS, and R221K N394K mutations in SpCas9 previously shown to improve Cas9 nuclease activity.

[0206] PEmax is described in Chen et al. (Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635-5652. e29 (2021)).

[0207] In a particular embodiment, the prime editor is PE6, in particular selected in the group consisting of PE6a, PE6b, PE6c, PE6d, PE6e, PE6f and PE6g, more particularly PE6b, PE6c or PE6d, even more particularly PE6b.

[0208] PE6a to PE6g are described in detail in Doman et al. (Cell. 2023 Aug 31; 186(18): 3983- 4002. e26., doi: 10.1016 / j .cell.2023.07.039). According to a particular embodiment, when the prime editor is PEmax, the cleavable linker of the fusion protein is localized in C-ter of the fusion protein, and contains, from N-ter to C-ter, a single protease cleavage site and at least one NES, in particular three NES.

[0209] In another embodiment, when the prime editor is PEmax, the fusion protein may comprise two cleavable linkers: a first cleavable linker localized between the virus-derived assembly protein and the prime editor, and a second cleavable linker localized in C-ter of the prime editor. In particular, the first cleavable linker contains, from N-ter to C-ter, a first protease cleavage site, at least one NES, in particular three NES, and a second protease cleavage site, and the second cleavable linker contains, from N-ter to C-ter, a single protease cleavage site and at least one NES, in particular three NES.

[0210] According to a particular embodiment, when the prime editor is PEmax or PE6, in particular PE6b, PE6c or PE6d, the cleavable linker of the fusion protein is localized in C-ter of the fusion protein, and contains, from N-ter to C-ter, a single protease cleavage site and at least one NES, in particular three NES.

[0211] In another embodiment, when the prime editor is PEmax or PE6, in particular PE6b, PE6c or PE6d, the fusion protein may comprise two cleavable linkers: a first cleavable linker localized between the virus-derived assembly protein and the prime editor, and a second cleavable linker localized in C-ter of the prime editor. In particular, the first cleavable linker contains, from N-ter to C-ter, a first protease cleavage site, at least one NES, in particular three NES, and a second protease cleavage site and the second cleavable linker contains, from N-ter to C-ter, a single protease cleavage site and at least one NES, in particular three NES.

[0212] As it has been demonstrated by the present inventors, a fusion protein of the invention is successfully integrated within the resulting virus-derived particle and the Prime editor moiety is fully active, i.e. possesses its endonuclease activity. According to those embodiments, the embedded Prime Editor is released inside the target cells following the entering of the virus- derived particles.

[0213] According to these other embodiments, the said fusion protein is integrated within the resulting virus-derived particle at the time of its production by the producing cells. Then, part or all of the said fusion proteins may be cleaved in the final virus-derived particles, leading to a population of virus-derived particles comprising (i) a part of the virus-derived particles wherein none the said fusion protein has been cleaved, (ii) a part of the virus-derived particles wherein at least a part of the said fusion proteins have been cleaved, leading the release of Prime Editor moiety inside the virus-derived particles and (iii) a part of the virus-derived particles wherein all or almost all of the said fusion proteins have been cleaved, leading the release of all or almost all of the Prime Editor moieties inside the virus-derived particles.

[0214] It shall be understood that a virus-derived particle as used herein is produced in packaging cells that notably express a protein between (i) a viral derived assembly protein, (ii) a cleavable linker and (iii) a Prime Editor, in the form of a fusion protein. The fusion protein is incorporated as such in the virus-derived particle and is then at least partly cleaved in the virus-derived particle so as to release the different moieties of the protein, which are functional in the virus-derived particle as it is shown in the examples herein.

[0215] However, because the Prime Editor is initially incorporated in the virus-derived particles under the form of the said fusion protein, there are a number of intermediate states wherein the Prime Editor is partly present under the form of the fusion protein and partly present as a free Prime Editor resulting from the cleavage of the fusion protein. pegRNA

[0216] A virus-derived particle according to the invention may further comprise a prime editing guide RNA, also known as a pegRNA or PEgRNA. The pegRNA are required to generate a site-directed alteration in a target nucleic acid, when using a virus-derived particle as described herein.

[0217] The pegRNA may be included in the VLP according to the invention or introduced by other means in the target cell containing the target gene to be edited.

[0218] A pegRNA according to the present invention may comprise two stem-loop structures in 3’. By “in 3 ’ of the pegRNA”, it is understood that the 3 ’ extremity of the pegRNA is downstream from the fixed elements of the pegRNA, i.e. downstream of the structures which are usually present in pegRNAs and are recognized by a Cas9. These structures may be for instance an ST2 loop and a tetraloop. The structures in 3’ of the pegRNA according to the invention are therefore different from these fixed elements present in the pegRNA, in particular are different from an ST2 loop and a tetraloop. In other words, the structures in 3 ’ of the pegRNA correspond to variable elements of the pegRNA, which are not always present in pegRNAs that are recognized by a Cas9. A stem-loop is an intramolecular base pairing that can occur in single stranded DNA or RNA if sequences of two regions of the same strand are complementary to each other. Stem-loop structures are well known in the art.

[0219] In a particular embodiment, the two stem-loop structures of the pegRNA are selected from trimmedEvoPreQi (herein after also referred to as tEvoPreQi or tEvQl), CSY4, MS2, TAR, PP7, Box C / D, Box B or the histone mRNA stem loop. In particular, the two stem-loop structures are selected from tEvoPreQi, CSY4, TAR and PP7. In a particular embodiment, when the prime editor of the fusion protein is PE6, the two stem-loop structures are CSY4 and / or TAR, in particular CSY4 and TAR.

[0220] The number of Prime Editing guide RNAs, which may also be termed “PE guide RNAs” or “pegRNAs”, may vary depending of the kind of alteration(s) to the target nucleic acids which is(are) sought. A single pegRNA may be used in combination with a virus-derived particle for generating a single DNA cleavage event in the target nucleic acid. Two or more pegRNAs may be used in combination with a virus-derived particle for generating two or more cleavage events in the target nucleic acids, or alternatively to generate cleavage event(s) in a plurality of target nucleic acids. Each guide RNA hybridizes with a specific target sequence comprised in a target nucleic acid.

[0221] Methods for designing guide RNAs that, when combined with a Prime Editor, generate the cleavage of a target nucleic acid, are well known from the one skilled in the art. As it is well known in the art, a guide RNA is a polynucleotide having sufficient complementarity with a target nucleic acid to hybridize with the said target nucleic acid and direct sequence-specific binding of a Prime Editor complex to the said target nucleic acid.

[0222] Various tools are readily available to the one skilled in the art for designing guide RNAs. Guide RNAs may for example be designed according to the teachings of Ran et al. (2013, Cell, Vol. 154 : 1380-1389), Mail et al. (2013, Science, Vol. 339 : 823-826), Wang et al. (2013, Cell, Vol. 153 : 910-918), Jao et al. (2013, Proc Natl Acad Sic USA, Vol. 110 : 13904-13909), Cong et al. (2013, Science, Vol. 339 : 819-823), Shalem et al. (2014, Science, Vol. 343 : 84-87), Maeder et al. (2013, Nat Methods ; Vol. 10 : 977-979), Qi et al. (2013, Cell, Vol. 152 : 1173-1183), Farboud et al. (2015, Genetics, doi 10.1534 / genetics.l l5.175166) or Ma et al. (2013, BioMed research International, Vol. 2013, Article ID 270805, doi.org / 10.1155 / 2013 / 270805). In particular, various tools are available to one skilled in the art for designing pegRNAs, such as according the teachings of Anzalone et al. (Search-and-replace genome editing without doublestrand breaks or donor DNA. Nature. 2019;576(7785):149-157. Doi:10.1038 / s41586-019-1711- 4), Kim et al. (Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol. 2021 ;39(2) : 198-206. Doi :10.1038 / s41587-020-0677-y), Morris et al. (Automated design of CRISPR prime editors for 56,000 human pathogenic variants. iScience. 2021;24(ll):103380. Published 2021 Oct 30. Doi:10.1016 / j.isci.2021.103380), Koeppel et al., (Predicting efficiency of writing short sequences into the genome using prime editing, 2021.11.10.468024; doi: https: / / doi.org / 10.1101 / 2021.l l.10.468024), Li et al. (Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat Commun. 2022;13(l): 1669. Published 2022 Mar 29. Doi : 10.1038 / s41467-022-29339-9), Liu et al. (Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Res. 2021 ;31 (10): 1134- 1136. Doi: 10.1038 / s41422-021-00520-x), Hsu et al. (PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat Commun. 2021;12(l):1034. Published 2021 Feb 15. Doi: 10.1038 / s41467-021-21337-7), Anderson et al. (pegIT - a web-based design tool for prime editing. Nucleic Acids Res. 2021;49(Wl):W505-W509. Doi:10.1093 / nar / gkab427), Hwang et al. (PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res. 2021;49(Wl):W499-W504. Doi:10.1093 / nar / gkab319), Mathis et al. (Predicting prime editing efficiency and product purity by deep learning. Nat Biotechnol 41, 1151-1159 (2023). https : / / doi.org / 10.1038 / s41587-022-01613-7) and Doman et al. (Designing and executing prime editing experiments in mammalian cells. Nature protocols vol. 17,11 (2022): 2431 -2468. Doi: 10.1038 / s41596-022-00724-4).

[0223] The one or more pegRNAs are comprised inside the virus-derived particle. Typically, the virus- derived particles are produced by packaging cells expressing the fusion proteins described above and the one or more pegRNAs. According to these embodiments, the one or more pegRNAs are incorporated within the virus-derived particles while these are produced by the packaging cells.

[0224] In a particular embodiment, transcription of the pegRNA is ensured by type 3 Pol III promoters particularly dedicated to the transcription of small non-coding RNAs, including 5S rRNA (type 1), tRNAs (type 2), and other essential RNAs (type 3) such as the U6 snRNA. The use of the POL III U6 promoter drives expression of pegRNAs in the VLP producing cells.

[0225] According to a particular embodiment, transcription of the pegRNA is ensured by a Pol II promoter. In a particular embodiment, transcription is ensured by a Pol II CMV promoter. In particular, a pegRNA according to the invention may be obtained from a cassette that encodes a pegRNA in an intronic sequence under the control of a Pol II promoter, in particular of a Pol II CMV promoter.

[0226] In a particular embodiment, the pegRNA encoded in the cassette is flanked on either side by a nuclease cleaving site. Nuclease cleaving sites are known in the art and further described above. Such sites include csy4 and cas6 nuclease cleaving sites. In particular, the cas6 nuclease is the Cas6f nuclease, otherwise known as Csy4.

[0227] In a particular embodiment, the pegRNA encoded in the cassette is flanked on either side by a csy4 nuclease cleaving site (csy4 or cas6 is a endonuclease specific to a RNA hairpin).

[0228] Upon expression of the Csy4 protein, which cleaves in 3’ of the Csy4 RNA stem loop, the pegRNA is released from the intron. A csy4-sl structure remains in the 3’ extremity of the pegRNA after cleavage.

[0229] In a particular embodiment, the one or more pegRNA(s) are in the form of ribonucleoprotein complexes which are complexes of the Prime Editor with a pegRNA.

[0230] In these embodiments, the virus-derived particles comprise one or more kinds of complexes of a Prime Editor and a pegRNA, wherein each complex comprise a single Prime Editor complexed with a single pegRNA. In some of these embodiments wherein a plurality of cleavages of a target nucleic acid is sought, the virus-derived particles comprise the same number of kinds of complexes, each kind of complex being specific for generating a DNA cleavage at a desired location of a target nucleic acid to which the corresponding pegRNA hybridize.

[0231] According to a particular embodiment, the one or more pegRNAs are initially produced by specific packaging cells expressing the said one or more pegRNAs and also expressing the viral protein(s) which are required for producing other viral particles or other viral vesicles (or other Virus-Like Particles or VLPs). Then, the pegRNA(s)-containing viral particles are brought into contact with a virus-derived particle comprising a Prime Editor, so as to generate, by complementation, the final virus-derived particles comprising both a Prime Editor and the one or more pegRNAs that were initially contained in the said other viral particles. Illustratively, Gagbased Virus-derived particles comprising a Prime Editor which are described herein may be brought into contact with VSV-G-based viral particles comprising one or more pegRNAs, so as to obtain final virus-derived particles comprising the Prime Editor and the one or more pegRNAs and wherein the said final virus-derived particles consist of VSV-G pseudotyped Gag-based VLPs.

[0232] In some other embodiments part of all of the said one or more guide RNAs are not comprised inside the virus-derived particles but are instead complexed with these virus-derived particles. According to these other embodiments, the guide RNAs which are complexed with the virus- derived particles also enter into the target cells with the virus-derived particles to which these guide RNAs are complexed.

[0233] In a particular embodiment, a VLP according to the invention comprises:

[0234] - a fusion protein containing, from N-ter to C-ter, a virus-derived assembly protein, a prime editor and a cleavable linker,

[0235] - a prime editor that is PEmax, and

[0236] - a pegRNA comprising two stem-loop structures in 3’, in particular selected from trimmedEvoPreQi, CSY4, TAR and PP7.

[0237] In a particular embodiment, a VLP according to the invention comprises:

[0238] - a fusion protein containing, from N-ter to C-ter, a virus-derived assembly protein, a prime editor and a cleavable linker,

[0239] - a prime editor that is PEmax or PE6, in particular PE6b, PE6c or PE6d, even more particularly PE6b, and

[0240] - a pegRNA comprising two stem-loop structures in 3’ selected from trimmedEvoPreQi, CSY4, TAR and PP7.

[0241] In a particular embodiment, a VLP according to the invention comprises:

[0242] - a fusion protein containing, from N-ter to C-ter, a virus-derived assembly protein, a prime editor and a cleavable linker,

[0243] - a prime editor that is PEmax or PE6, in particular PE6b, PE6c or PE6d, even more particularly PE6b, and

[0244] - a pegRNA comprising two stem-loop structures in 3’, in particular CSY4 and TAR. In a particular embodiment, a VLP according to the invention comprises:

[0245] - a fusion protein containing, from N-ter to C-ter, a virus-derived assembly protein, a prime editor and a cleavable linker containing a first protease cleavage site (PS) and at least one nuclear export signal (NES), in particular three NES,

[0246] - a prime editor that is PEmax, and

[0247] - a pegRNA comprising two stem-loop structures in 3’, in particular selected from trimmedEvoPreQi, CSY4, TAR and PP7.

[0248] In a particular embodiment, a VLP according to the invention comprises:

[0249] - a fusion protein containing, from N-ter to C-ter, a virus-derived assembly protein, a prime editor and a cleavable linker containing a first protease cleavage site (PS) and at least one nuclear export signal (NES), in particular three NES,

[0250] - a prime editor that is PEmax or PE6, in particular PE6b, PE6c or PE6d, even more particularly PE6b, and

[0251] - a pegRNA comprising two stem-loop structures in 3’, in particular selected from trimmedEvoPreQi, CSY4, TAR and PP7.

[0252] In a particular embodiment, a VLP according to the invention comprises:

[0253] - a fusion protein containing, from N-ter to C-ter, a virus-derived assembly protein, a first cleavable linker, a prime editor and a second cleavable linker, the first cleavable linker containing a first protease cleavage site, at least one NES, in particular three NES, and a second protease cleavage site and the second cleavable linker containing a first protease cleavage site (PS) and at least one nuclear export signal (NES), in particular three NES,

[0254] - a prime editor that is PEmax or PE6, in particular PE6b, PE6c or PE6d, even more particularly PE6b, and

[0255] - a pegRNA comprising two stem-loop structures in 3’, in particular selected from trimmedEvoPreQi, CSY4, TAR and PP7.

[0256] A virus-derived particle of the invention may further comprise one or more target molecules. In a particular embodiment, a VLP further comprises at least one target molecule that binds to pegRNA. In particular, the VLP comprises at least one target molecule that binds to one or more of the stem-loop structures of the pegRNA.

[0257] In a particular embodiment, the at least one target molecule is selected from the group consisting of MCP, PP7CP, TAT, Csy4 (wild type or H29A mutant), L7Ae, LambdaN, SLBP and Com, in particular the target molecule is a PP7CP or is a TAT.

[0258] A virus-derived particle of the invention may further comprise one or more additional protein(s).

[0259] In particular, a VLP of the invention may comprise one or more proteins selected from reporter proteins, from helper proteins, and from DNA and / or RNA binding proteins.

[0260] The additional protein(s) may be selected from fluorescent proteins such as mCherry, GFP, mKeita or luciferase; transcription factors ; proteins limiting the expression of a surface cell marker in recipient cells; proteins boosting expression of a cell surface marker in recipient cells; proteins generating a selectable phenotype such as proteins inducing a resistance to a drug; recombinases such as CRE; integrases such as BxBl; internal proteins facilitating the oncorporation of specific fusogens or viral envelopes; helper proteins including hMLHldn, P53dd, P65, Rad51, T5 exonuclease and FEN1 or binding proteins including MS2 coat protein, PP7 Coat protein, and TATI from HIV.

[0261] Auxiliary nucleic acids

[0262] For the purpose of altering a target nucleic acid by using virus-derived particles as described herein, and depending on the prime editor system that is used, it may further be made use of an auxiliary nucleic acid in combination with these virus-derived particles.

[0263] According to a particular embodiment, a VLP may further comprise one or more auxiliary nucleic acids.

[0264] An auxiliary nucleic acid, as described herein, is a nucleic acid that assists the prime editor in the integration of a specific sequence at a specific localization.

[0265] In particular, the auxiliary nucleic acid may be a template nucleic acid.

[0266] In a particular embodiment, the auxiliary nucleic acid may be a circular DNA. Prime Editing technologies, except GRANDE, use PE3 as prime editor to add integrase recognition sites in DNA (eg attP) then an auxiliary nucleic acid containing recombination sites is integrated into the genome by an integrase. The integrase can be added in cis as it can be fused to the MMLV-RT (PASTE and Click Editing) or in trans by adding it during prime editing events (TWIN-PE).

[0267] Examples of auxiliary nucleic acids include nicking gRNAs (ngRNA) and single guide RNAs (sgRNA). Such nucleic acids are described in Anzalone et al. (Nature volume 576, pages 149-157 (2019)).

[0268] In a particular embodiment, the auxiliary nucleic acid comprises at least one recombination site.

[0269] In particular, an auxiliary nucleic acid may be required when the prime editing method is selected from PASTE, TWIN-PE, Click Editing and GRANDE methods, in particular is the PASTE method.

[0270] PASTE is programmable addition via site-specific targeting elements (PASTE).

[0271] The auxiliary nucleic acid, or template nucleic acid, may have any suitable length, such as about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 2000, 5000, 10 000, 25 000, 50 000 or more nucleotides in length.

[0272] Based on the general knowledge from the one skilled in the art, practically the sole requirement for designing an auxiliary nucleic acid for the purpose of homologous recombination is the prior knowledge of the nucleic sequence of the target nucleic acid.

[0273] In some other embodiments, an auxiliary nucleic acid is not comprised inside the virus-derived particle but is complexed to the virus-derived particles.

[0274] Plurality of nucleotides

[0275] The present invention further relates to a plurality of polynucleotides comprising:

[0276] (i) one or more polynucleotide(s) encoding a fusion protein comprising: a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity;

[0277] (ii) a polynucleotide encoding a prime editing guide RNA (pegRNA), in particular a pegRNA bound to the napDNAbp, and

[0278] (iii) optionally a polynucleotide encoding one or more pseudotyping viral envelope protein(s).

[0279] A polynucleotide is composed of nucleotide monomers covalently bonded in a chain. Herein, it is synonymous with a nucleic acid, or a nucleic acid sequence.

[0280] Such a plurality of polynucleotides may serve to produce VLPs according to the invention.

[0281] In particular, a method for producing the presently described VLPs comprises transfecting, transducing, electroporating, or otherwise inserting into a cell line a plurality of polynucleotides that together encode all the components of the VLPs.

[0282] In some embodiments, the present disclosure provides one or more vectors comprising one, two or three of the plurality of polynucleotides provided herein. In certain embodiments, each of the polynucleotides are on separate vectors. In certain embodiments, one or more of the first, second, and third polynucleotides are on the same vector.

[0283] The plurality of nucleotides comprises one or more nucleotides encoding a fusion protein comprising a virus-derived assembly protein, cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity.

[0284] The different components of the fusion protein may be provided as a single polynucleotide sequence, or as separate polynucleotide sequences. In a particular embodiment, the different components of the fusion protein are provided as a single polynucleotide sequence. In a particular embodiment, the plurality of polynucleotides comprises:

[0285] (i) a first polynucleotide encoding a fusion protein comprising: a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity;

[0286] (ii) a second polynucleotide encoding a prime editing guide RNA (pegRNA), in particular a pegRNA bound to the napDNAbp, and

[0287] (iii) optionally a third polynucleotide encoding one or more pseudotyping viral envelope protein(s).

[0288] Virus-derived assembly proteins, cleavable linkers and prime editors encoded by the polynucleotides are described further above.

[0289] In a particular embodiment, the polynucleotide encoding a cleavable linker contains, from 5’ to 3’ a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS). In particular, the polynucleotide encoding a cleavable linker contains, from 5’ to 3’ a first protease cleavage site (PS), three nuclear export signals (NES) and a second protease cleavage site (PS).

[0290] In another embodiment, the polynucleotide encoding a cleavable linker contains, from 5’ to 3’, a first protease cleavage site (PS) and at least one nuclear export signal (NES). In particular, the polynucleotide encoding a cleavable linker contains, from 5’ to 3’, a first protease cleavage site (PS) and three nuclear export signals (NES).

[0291] In a particular embodiment, the NES has a nucleic acid sequence of SEQ ID NO: 14 (CTGCCTCCACTTGAAAGACTGACACTG) (NESI), SEQ ID NO: 76 (TTACCTCCTTTAGAACGATTAACACTC) (NES2) or of SEQ ID NO: 15 (CTTCCGCCTCTTGAGAGATTGACATTA) (NES3). Further, pegRNAs suitable according to the present invention, included in the plurality of polynucleotides herein, are described above. According to another embodiment, the first polynucleotide encoding a fusion protein further comprises a second cleavable linker. The first and second cleavable linkers suitable to the present invention are described above. According to this embodiment, the polynucleotide encoding the fusion protein comprises, from 5’ to 3’, nucleic acids encoding a virus-derived assembly protein, a first cleavable linker, a prime editor as described above, and a second cleavable linker, wherein the first and second cleavable linkers contain from 5’ to 3’, a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS), in particular a first protease cleavage site (PS), three nuclear export signals (NES) and optionally a second protease cleavage site (PS).

[0292] In particular, the pegRNA is contained in an intron under the control of strong promoters, in particular strong human promoters, driving the expression of essential human genes. Such promoters include Pol II, CAG and EFl promoter.

[0293] In a particular embodiment, the pegRNA is contained in an intron under the control of a Pol II promoter. In a particular embodiment, the pegRNA is contained in an intron under the control of a Pol II CMV promoter.

[0294] According to a particular embodiment, the pegRNA is flanked on either side by a nuclease cleaving site.

[0295] Nuclease cleaving sites are well known in the art and have been described further above.

[0296] In a particular embodiment, the pegRNA is flanked on either side by a nuclease cleaving site by a csy4 endonuclease cleaving site.

[0297] According to a particular object, the present invention further relates to a method for producing a virus-derived particle comprising at least the steps of: providing the plurality of polynucleotides as described herein ; and providing a nuclease, in particular a Cas6 nuclease, more particularly Cas6f nuclease.

[0298] Methods for producing VLPs are known to one skilled in the art. Such methods are also referred to elsewhere in the specification.

[0299] In a particular embodiment, the method for producing a VLP according to the invention can be carried out in a packaging cell as described below. Thus, the present invention further relates to a method for producing a virus-derived particle comprising at least the steps of:

[0300] - culturing a packaging cell comprising:

[0301] - the plurality of polynucleotides as described herein; and

[0302] - a nuclease, in particular a Cas6 nuclease, more particularly Cas6f nuclease; and

[0303] - recovering and purifying the virus-derived particle produced from said packaging cell.

[0304] Nucleic acid expression vectors

[0305] As already stated elsewhere in the present specification, a virus-derived particle as described herein is produced in cells, also named packaging cells herein, which express the required proteins, i.e. at least a fusion protein and one or more viral proteins required for forming the viral particles, which may also be termed Virus-Like Particles or VLPs. In particular embodiments, the packaging cells also express one or more PE guide RNAs and, when necessary.

[0306] The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in eukaryotic cells generally comprise promoters, enhancers, and termination and polyadenylation signals. In some embodiments, “expression vectors” are used in order to permit pseudotyping of the viral envelope proteins.

[0307] Generally, vectors for expressing the required proteins or nucleic acids are vectors suitable for expressing nucleic sequences within the desired host cells that are used as packaging cells. Preferably, the packaging cells are insect cells or mammalian cells. Notably, vectors for expressing the required proteins or nucleic acids comprise an open reading frame which is placed under the control of regulatory elements that are functional in the packaging cell wherein their expression is sought. Notably, these vectors comprise, for each protein or nucleic acid to be expressed, an open reading frame which is placed under the control of a suitable promoter sequence, as well as optionally a polyadenylation sequence.

[0308] The packaging cell line provides the viral proteins required for particle assembly (Markowitz et al., 1988, J. Virol., Vol. 62 : 1120). As it is well known in the art, a nucleic acid vector is introduced into the packaging cell by any of a variety of techniques (e.g., calcium phosphate co-precipitation, lipofection, electroporation). The viral proteins produced by the packaging cell mediate the insertion of the viral protein(s) and of the fusion protein into virus-derived particles, which are then released into the culture supernatant.

[0309] The nucleic acid vectors used may be derived from a retrovirus (e.g., a lentivirus). Retrovirus vectors suitable for producing the virus-derived particles described herein allow (1) transfection of the packaging vectors and envelope vectors into the host cell to form a packaging cell line that produces the virus-derived particles essentially free from packaging vector RNA, and (2) the packaging of the fusion protein and optionally also of PE guide RNA(s).

[0310] Vectors and packaging cells for use according to the present invention are illustrated in the examples herein.

[0311] Illustratively, a vector for expressing the viral structural protein / fusion protein, e.g. a GAG-PE- LINKER, may be prepared by the one skilled in the art as taught by Voelkel et al. (2010, Proc Natl Acad Sci USA, Vol. 107: 7805-7810).

[0312] Illustratively, a vector for expressing the viral structural protein, e.g. a Gag protein or a Gag-Pro- Pol fusion protein, and optionally also a viral envelope protein, e.g. a VS V-G protein or a BAEV- G protein, may be prepared by the one skilled in the art according to the teachings of Negre et al. (2000, Gene Ther, Vol. 7: 1613-1623) and of Yee et al. (1994, Methids Cell Biol, Vol. 43 PtA: 99-112).

[0313] Packaging cells

[0314] The present invention provides a cell line for producing a virus-derived particle according to the invention, in particular comprising a plurality of polynucleotides as described above.

[0315] The cell line or host cell is a cell into which a vector of interest may be introduced and wherein it may be replicated, and, in the case of an expression vector, in which one or more vector-based genes may be expressed.

[0316] Any suitable permissive or packaging cell known in the art may be employed in the production of the virus-derived particles described herein. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of the virus-derived particles in the practice of the invention include, for example, human cell lines, such as VERO, WI38, MRC5, A549, HEK293, HEK293T, B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines. Illustrative cell lines for use as packaging cells are insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present invention. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. Cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred insect cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings concerning use of insect cells for expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995 ); O’Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994 ) ; Samulski et al., J. Vir. 63 :3822-8 (1989 ) ; Kajigaya et al., Proc. Nat’l. Acad. Sci. USA 88 : 4646-50 (1991 ) ; Ruffing et al., J. Vir. 66 :6922-30 (1992 ) ; Kimbauer et al., Vir. 219 :37-44 (1996 ) ; Zhao et al., Vir. 272 :382-93 (2000 ) ; and Samulski et al., U.S. Pat. No. 6,204,059 .

[0317] The cells may be supplied with any one or more of the stated functions already incorporated, e.g., a cell line with one or more vector functions incorporated extra-chromosomally or integrated into the cell’s chromosomal DNA, a cell line with one or more packaging functions incorporated extra-chromosomally or integrated into the cell’s chromosomal DNA, or a cell line with helper functions incorporated extra-chromosomally or integrated into the cell’s chromosomal DNA. A packaging cell line is a suitable host cell transfected by one or more nucleic acid vectors that, under achievable conditions, produces virus-derived particles comprising a Cas protein and, in some embodiments, also one or more CRIPSR guide RNA(s) and eventually also a targeting nucleic acid.

[0318] As used herein, the term “packaging cell lines” is typically used in reference to cell lines that express viral structural proteins (e.g., gag, pol and env), but do not contain a packaging signal. For example, a cell line has been genetically engineered to carry at one chromosomal site within its genome, a 5’-LTR-gag-pol-3’-LTR fragment that lacks a functional psi+ sequence (designated as A-psi), and a 5’-LTR-env-3’-LTR fragment that is also A-psi located at another chromosomal site.

[0319] A number of cell types can be used, which encompasses: a) NIH-3T3 murine cells which are currently widely used as packaging cells producing recombinant retroviruses in clinical use (Takahara et al., Journal of Virology, (June 1992), 66 (6) 3725-32). b) TK' cell lines have already been described, including NIH-3T3 TK cells (F. Wagner et al., EMBO Journal (1985), Vol. 4 (n°3): 663-666); these cells can be killed when they are cultivated in selective culture media such as HAT. If they are complemented for the kinase thymidine function, for example those from the HSV1-TK virus, they can grow in a selective medium; such lines thus offer the possibility of using the HSV1-TK gene as a selection gene. The gene coding for the thymidine kinase of HSV1 or one of its functional derivatives is also widely used as a transgene as a pro-drug transforming ganciclovir or acyclovir into a drug which is cytotoxic for the cell, and it can thus be applied to selective cell destruction, for example of cancerous cells (see, for example, International patent application WO 95 / 22617).

[0320] Illustratively, the packaging cells may be the well-known HEK293T cell line, as shown in the examples herein.

[0321] The present invention also relates to a cell line for producing a virus-derived particle as described herein, comprising:

[0322] - one or more polynucleotides encoding the proteins required for forming the said virus- derived particle, and

[0323] - a polynucleotide comprising an expression cassette encoding a viral structural protein, a cleavable linker and a prime editor.

[0324] In some embodiments, a polynucleotide encoding a protein required for forming the said virus- derived particle encompasses a nucleic acid encoding a viral structural protein, such as a Gag protein.

[0325] In some embodiments, the said cell line also comprises a polynucleotide encoding a viral envelope protein, such as a viral envelope protein selected in a group comprising a VSV-G protein and a BAEV-G protein.

[0326] In some embodiments, the said cell line further comprises polynucleotide(s) encoding one or more PE guide RNA(s).

[0327] Compositions and kits

[0328] The present invention provides virus-derived particles compositions and kits suitable for use in therapy (in vivo or ex vivo) that are described herein. The present invention relates to a composition for altering a target nucleic acid in a eukaryotic cell, which composition comprises at least one virus-derived particle as described in the present specification.

[0329] In some embodiments, the said composition further comprises one or more PE guide RNA(s) (or pegRNAs or PEgRNAs).

[0330] In some of these embodiments, the said one or more PE guide RNA(s) is(are) comprised in virus- derived particles.

[0331] In some other embodiments, the said one or more PE guide RNA(s) is(are) complexed with the said virus-derived particles.

[0332] According to some of these embodiments, each PEgRNA present in the composition is comprised in a specific kind of vesicles. According to some other of these embodiments, more than one PEgRNA, which includes all PEgRNA(s), are comprised in a specific kind of vesicles.

[0333] A “specific kind” of vesicle, as used herein is defined uniquely as regards its specific content in PEgRNA(s) irrespective of the structural features of the vesicle itself.

[0334] Most preferably, the said vesicles are comprised of viral proteins. In some embodiments, the said vesicles have the same structural features of viral proteins as the virus-derived particles containing a Prime Edtor that are described in the present specification. In some other embodiments, the said vesicles are mainly or fully composed of viral envelope proteins, such as, for example, VSV-G or BAEV-G.

[0335] When present in a composition according to the invention, the PE-containing virus-derived particles and the PEgRNA(s) - containing vesicles trans-complement so as to efficiently generate the desired nucleic acid alteration(s) in the target cells. Such a trans-complementation in another technical context is taught by Mangeot et al. (2011, Ther J am Soc Gene Ther, Vol. 19: 1656- 1666).

[0336] Compositions as described herein encompass pharmaceutical compositions that are used for the purpose of performing a method of gene therapy in mammals in need thereof, which includes nonhuman mammals and human individuals in need thereof.

[0337] Compositions of the invention may be formulated for delivery to animals for veterinary purposes (e.g., livestock such as cattle, pigs, etc), and other non-human mammalian subjects, as well as to human subjects. The virus-derived particles may be formulated with a physiologically acceptable carrier for use in gene transfer and gene therapy applications.

[0338] In some embodiments, the said composition further comprises one or more transduction helper compounds. The transduction helper compounds are preferably selected in a group comprising cationic polymers, as described notably by Zuris et al. (2015, Nat Biotechnol, Vol. 33(n°l): 73- 80). The transduction helper compound may be selected in a group comprising polybrene (that may be also termed hexadimethrine bromide), protamine sulfate, 12-myristate 13 -acetate (also termed phorbol myristate acetate or PMA, as described by Johnston et al., 2014, Gene Th er, Vol. 21(12): 1008-1020), vectofusin (as described by Fenard et al., 2013, Molecular Therapy Nucleic Acids, Vol. 2: e90), poloxamer P338 (as described by Anastasov et al., 2016, Lentiviral vectors and exosomes as gene and protein delivery tools, in Methods in Molecular Biology, Vol. 1448: 49-61), RetroNectin® Reagent (commercialized by Clontech Laboratories Inc.), Viral Plus ® transduction enhancer (commercialized by Applied Biological Materials Inc.), TransPlus® Virus Transduction Enhancer(commercialized by Clinisciences), Lentiboost® (commercialized by Sirion Biotech), or ExpressMag® Transduction System (commercialized by Sigma-Aldrich). As shown in the examples herein, the said cationic transduction helper compound may consist of polybrene.

[0339] The compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Liquid preparations of the virus-derived particles compositions may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts. Alternatively, the compositions may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compositions of the invention may be administered to a subject at therapeutically effective doses to generate the desired genome alteration in a target nucleic acid contained in a target cell, in a target tissue or organ or in a target organism, particularly a target mammal, which encompasses a target non-human mammal and a human individual. A therapeutically effective dose refers to an amount of the composition sufficient to result in amelioration of symptoms caused by the occurrence of the desired genome alteration event in the target nucleic acid.

[0340] In an embodiment, an amount of composition of the invention is administered at a dose unit that is in the range of about 0.1-5 micrograms (pg) / ki logram (kg). To this end, a composition of the invention may be formulated in doses in the range of about 7 mg to about 350 mg to treat to treat an average subject of 70 kg in body weight. The amount of composition of the invention that may be administered may be selected in a group comprising 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg or 5.0 mg / kg. The dose of virus-derived particles in a unit dosage of the composition may be selected in a group comprising 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, or 750 mg, especially for treating an average subject of 70 kg in body weight. These doses can be given once or repeatedly, such as daily, every other day, weekly, biweekly, or monthly. In some embodiments, a virus- derived particles composition may be administered to a subject in one dose, or in two doses, or in three doses, or in four doses, or in five doses, or in six doses or more. The interval between dosages may be determined based the practitioner’s determination that there is a need thereof.

[0341] The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0342] A virus-derived particles composition may be in liquid or solid form.

[0343] Kits

[0344] The present invention further relates to kits for preparing the virus-derived particles of the invention. This invention concerns a kit for preparing virus-derived particles for altering a target nucleic acid in a eukaryotic cell comprising:

[0345] - a nucleic acid comprising an expression cassette encoding a fusion protein as described above, and

[0346] - a nucleic acid comprising one or more expression cassette(s) encoding virus-like assembly protein(s),

[0347] In some embodiments, the said kit further comprises a nucleic acid comprising an expression cassette encoding a pseudotyping viral envelope protein.

[0348] In some embodiments of the said kit, the virus-derived assembly protein is a virus-derived Gag protein.

[0349] In a particular embodiment, the virus-derived assembly protein is a virus-derived fragment of the GAG protein. GAG is a composite protein with Matrix-Capsid-Nucleocapsid (MACANC). A fragment of the GAG protein includes the MA-CA fragment, the MA fragment and the MA-NC fragment.

[0350] In some embodiments, the said Gag protein is encoded by an expression cassette selected in a group comprising an expression cassette encoding a GAG-PRO-POL polyprotein and an expression cassette encoding a GAG protein. In another embodiment, the said Gag protein is encoded by an expression cassette selected in a group comprising an expression cassette encoding a GAG-PRO or GAG-PRO-RT polyproteins and an expression cassette encoding a GAG protein.

[0351] In some embodiments, the said kit further comprises one or more nucleic acid(s) encoding a PE guide RNA

[0352] In certain embodiments of the said kit, the said nucleic acids are localized in an eukaryotic cell as a result of its transfection into the said eukaryotic cell. In some of these embodiments, the said nucleic acids are under the form of nucleic acid vectors in the said eukaryotic cells, which cells may also be termed packaging cells herein. In some other of these embodiments, part or all of these nucleic acids are integrated in the genome of these eukaryotic cells, which cells may also be termed packaging cells herein.

[0353] Thus, in some embodiments of a kit according to the invention, the said eukaryotic cell consists of a packaging cell line. The kits of the present invention may optionally comprise different containers (e.g., vial, ampoule, test tube, flask or bottle) for each individual composition or element comprised therein. The kit may contain additional reagents, such as buffers, diluents and the like, for formulation the individual components. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form.

[0354] Instructions for using the kit according to the methods described herein may be included. The instructional material may comprise a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the method of the invention in the kit for assessment of oocyte quality. A package insert may comprise text housed in any physical medium, e.g., paper, cardboard, film, or may be housed in an electronic medium such as a diskette, chip, memory stick or other electronic storage form. The instructional material of the kit of the invention may, for example, be affixed to a container which contains other contents of the kit, or be shipped together with a container which contains the kit. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the contents of the kit be used cooperatively by the recipient.

[0355] Methods for altering a target nucleic acid

[0356] The virus-derived particles as well as the compositions comprising them may be used for gene therapy.

[0357] A further aspect of the invention is a method of treating subjects with the virus-derived particles according to the invention or with compositions comprising them.

[0358] Administration of the virus-derived particles to a human subject or an animal in need thereof can be by any means known in the art for administering virus vectors.

[0359] Exemplary modes of administration include rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one may administer the virus in a local rather than systemic manner, for example, in a depot or sustained-release formulation. This invention also relates to a method for altering a target nucleic acid comprising at least a target sequence in an eukaryotic cell, comprising the steps of: a) bringing into contact the said eukaryotic cell with virus-derived particles as described herein, or with a composition as described in the present specification, and b) collecting the said eukaryotic cell having an altered target nucleic acid.

[0360] In some embodiments, the virus-derived particles, or compositions comprising them, are administered directly to the subject, in vivo. In some other embodiments, subject’s cells are provided, and then the said cells are transduced in vitro with the virus-derived particles, or with a composition comprising them. In a further method step, the transduced subject’s cells are administered back to the body of the subject.

[0361] In some embodiments, said method is performed in vitro or ex vivo.

[0362] The present invention also relates to a composition as described herein, for its use for preventing or treating any disease or disorder that is amenable to gene therapy.

[0363] The present invention provides for methods for preventing or treating any disease or disorder that is amenable to gene therapy. As used herein, “treatment” or “treating” refers to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, “treatment” or “treating” refers to an amelioration of at least one measurable physical parameter associated with a disease or disorder, not necessarily discernible by the subject. In yet another embodiment, “treatment” or “treating” refers to inhibiting the progression of a disease or disorder, either physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both. Other conditions, including cancer, immune disorders, and veterinary conditions, may also be treated. As used herein, “prevention” or “preventing” with respect to a disease or disorder relate to prophylactic treatment of the disease or the disorder, e.g., in an individual suspected to have the disease, or at risk for developing the disease. Prevention may include, but is not limited to, preventing or delaying onset or progression of the disease and / or maintaining one or more symptoms of the disease or disorder at a desired or sub- pathological level. The term “prevent” does not require the 100% elimination of the possibility or likelihood of occurrence of the event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of a composition or method as described herein. Types of diseases and disorders that can be prevented and / or treated by methods of the present invention include, but are not limited to, age-related macular degeneration; diabetic retinopathy; infectious diseases e.g., HIV pandemic flu, category 1 and 2 agents of biowarfare, or any new emerging viral infection; autoimmune diseases; cancer; multiple myeloma; diabetes; systemic lupus erythematosus (SLE); hepatitis C; multiple sclerosis; Alzheimer’s disease; parkinson’s disease; amyotrophic lateral sclerosis (ALS), huntington’s disease; epilepsy; chronic obstructive pulmonary disease (COPD); joint inflammation, arthritis; myocardial infarction (MI); congestive heart failure (CHF); hemophilia A; or hemophilia B.

[0364] Infectious diseases that can be treated or prevented by the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, helminths, and parasites. The invention is not limited to treating or preventing infectious diseases caused by intracellular pathogens. Many medically relevant microorganisms have been described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which are hereby incorporated herein by reference.

[0365] Types of cancers that can be treated or prevented by the methods of the present invention include, but are not limited to human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin’s disease and non-Hodgkin’s disease), multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease. The terms used in this specification generally have their ordinary meanings in the art. Certain terms are discussed elsewhere in the present disclosure, to provide additional guidance in describing the products and methods of the presently disclosed subject matter.

[0366] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0367] The terms “about” or “approximately” as used herein refer to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term “about” refers to ±10% of a given value. However, whenever the value in question refers to an indivisible object, such as a molecule or other object that would lose its identity once subdivided, then “about” refers to ±1 of the indivisible object.

[0368] It is understood that aspects and embodiments of the present disclosure described herein include “having,” “comprising,” “consisting of,” and “consisting essentially of’ aspects and embodiments. The words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of’ implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of’ implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic and novel characteristic(s) of the disclosure. It is understood that the different embodiments of the disclosure using the term “comprising” or equivalent cover the embodiments where this term is replaced with “consisting of’ or “consisting essentially of’.

[0369] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. The present invention is further illustrated, without being in any way limited to, the examples below.

[0370] EXAMPLES

[0371] Example 1: Delivery of Prime editing by prime editor virus-derived particles in human cells

[0372] In order to transfer prime editing tools (PE) by retroviral like particles (VLPs), the inventors fused a Cas9n-RT sequence to GAG from murine leukemia virus (MLV-GAG) and with GAG from human immunodeficiency virus (EHV-GAG), according to the following sequences:

[0373] These designs were previously validated to generate VLPs packing genome-engineering proteins (Hamilton, J. R. et al. Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Rep. 35, 109207 (2021), Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250-265. el6 (2022) and Mangeot, P. E. et al. Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins. Nat. Commun. 10, 45 (2019)).

[0374] The inventors investigated whether these constructs could produce VLPs able to package a model of pegRNA inserting three nucleotides in the HEK locus and edit recipient cells. They designed pegHEK3 having a sequence of SEQ ID NO: 18

[0375] (GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTG CTCAGTCTG).

[0376] VLPs were produced according to the protocol provided further below. The VLPs were used to edit HEK293T target cells.

[0377] In addition to MLV and HIV based VLPs, the inventors also produced VSV-G induced vesicles (no GAG fusion) since they could potentially efficiently package and deliver proteins. A method for producing such vesicles is described in Mangeot et al. (Mol Ther 2002). Briefly, the protein of interest is overexpressed in the producer cell in addition with VSVG: Particles are then produced that incorporate the protein of interest (passively).

[0378] The PCR assay detecting CTT insertion in the HEK3 locus is depicted in Figure 1. The corresponding image analysis is provided in the table below.

[0379] Table 1

[0380] This first assay revealed that MLV-based VLPs seemed to be the most efficient system to mediate PE, however HIV-based and VSVG-based VLPs were also efficient.

[0381] To facilitate measurement of editing, the inventors next constructed a reporter cassette encoding the YFP gene interrupted by a stop codon (YFPs) and a pegRNA specifically designed to correct the stop and restore YFP fluorescence. The SWIS or SWYS cassette had the sequence of SEQ ID NO: 19.

[0382] This cassette was inserted into a lentivector and multiple copies were stably integrated into HEK293T cells to generate an editing-reporter cell line that strongly expresses YFP once edited.

[0383] This particular system allowed the inventors to rapidly investigate different versions of YFPs- pegRNA and define the optimal size of the PBS regions. SWIS reporter cells were transfected by pegRNA modified to incorporate PBS of various sizes from 9 to 24 nucleotides in length. YFP conversion efficiencies were measured by FACS 72hours later (see Figure 2). The pegRNA had a sequence of SEQ ID NO: 20. Having defined an optimized pegRNA, the inventors next produced PE- VLPs aiming at correcting the YFPs and pseudotyped them with diverse viral envelopes.

[0384] PE delivery efficiencies measured for all pseudotypes are shown Figure 3.

[0385] The results of this assay demonstrate that the combined introduction of VSV-G, the R-less version of BAEV envelope (BRL), and human syncytin surpasses other multiplexing options, resulting in nearly a 2-fold increase in editing efficiencies in HEK293T reporter cells as compared with the VSVG / BRL tandem. Unless otherwise specified, this envelope triumvirat was chosen for the production of all VLPs in the results below.

[0386] Taken together these findings indicate that pseudotyped PE- VLPs can mediate detectable primeediting events in recipient cells, at the level of transduced integrated cassettes (YFPs) or within an endogenous locus (HEK3).

[0387] Example 2: Optimization of PE-reporter cell line

[0388] The inventors explored different optimizations of reagents involved in the PE process and improved the PE-reporter cell line.

[0389] Indeed, the SWIS cell line described in example 1 resulted from the transduction of HEK293T with a high mutilplicity of infection (moi) of SWIS lentivectors.

[0390] The inventors decided to generate a more physiological reporter cell-line harboring few, ideally one, copy of YFPs per genome. To achieve this goal the SWIS cassette was inserted into a lentivector equipped with the Puromycin resistance.

[0391] This SWISIP2 cassette includes the cassette from the 5’LTR to the 3’LTR of the lentivirus. The YFPs-IRES-Puro sequence, corresponding to the ORF coding sequence has the DNA sequence of SEQ ID NO: 21.

[0392] HEK293T were transduced at low moi (multiplicity of infection) (0,5) and a single-insertion population was selected and amplified. qPCR quantification of inserted copies revealed that YFPs copies within this new reporting system did not exceed 1. This approach was also followed to generate other reporter cell lines like A549 cells and U2OS cells.

[0393] Example 3: Prime editor evolutions

[0394] The inventors attempted to improve the GAG-PE construct VI (provided in example 1 under SEQ ID NO: 16) by elimination of the RNaseH domain included in the Reverse transcriptase sequence. This domain induces degradation of RNA during reverse transcription.

[0395] This strategy led to constructs GAG-PE V2.

[0396] Furthermore, the inventors tested and noted that insertion of nuclear exporting signals (NES) between GAG and PE improved production and delivery.

[0397] Consequently, the inventors designed an innovative NES-containing linker and inserted it in the GAG-PE VI construct, giving rise to GAG-PE V3. To create a 3XNES sequence, three primer pairs were designed and hybridized thus creating three complementary HIV1-NES (sequences taken from Banskota, Samagya et al. “Engineered viruslike particles for efficient in vivo delivery of therapeutic proteins.” Cell vol. 185,2 (2022): 250- 265. el6. doi: 10.1016 / j. cell.2021.12.021). The most 5’ NES contained a homology sequence to Notl restriction enzyme while the most 3’ NES contained a Xbal restriction enzyme homology sequence. Then, the 3xNES fragments were cloned into the vector pCMV-PE2 digested by Notl and Xbal. Finally, GAG were PCR amplified from GAG-PE VI and cloned into the SnaBI-Notl restriction enzyme sites. The 3 ’ protease site was cloned by annealing and ligation of a primer pair containing two Notl restriction enzyme sites into the vector GAG-3XNES-PE. This recombinant vector was named GAG-PE V3.

[0398] GAG-PE V4 corresponds to a particular design where the two improvements were combined: no RNaseH domain and presence of a NES linker between GAG and PE.

[0399] These new versions of GAG-PE were next assessed for their efficacy to produce YFPs correcting- PE VLPs and mediate editing. Three independent batches of VLPs were prepared for each condition and used to transduced single-copy YFPs reporter cells. Fold change relative to GAG- PE VI are given.

[0400] The results provided in Figure 4 indicate that both V3 and V4 were able to deliver two-fold more PE as compared with GAG-PE VI.

[0401] Example 4: Optimized Prime editor and pegRNA

[0402] As another effort regarding the optimization of PE components, the inventors also modified the pegRNA by addition of RNA structures to its 3’ terminus region. pegRNA correcting the YFPs cassette were engineered to incorporate diverse stabilizing structures between the PBS domain and a 3’ terminal trimmedEvoPreQi domain. Hairpins tested were the MS2 stem loop, the PP7 stem loop and the HIV-TAR domain. Three independent batches of PE- VLPs were produced with these modified epegRNAs. A classic epegRNA (that contains only trimmedEvoPreQi) and a non-modified pegRNA containing no specific 3 ’-structure were also tested as well as VLP produced without editor (Ctrl).

[0403] All VLP batches were next used to transduce YFPs-reporter cells and editing efficiencies were next monitored by FACS three days after transduction. Transduction assays were performed in triplicate into 10000-recipient cells using 5ul of concentrated VLPs.

[0404] Results are given as the means of % of YFP cells measured after transduction in Figure 5. Addition of any hairpin upstream the trimmedEvoPreQi domain systematically improved PE- VLP performance. Further, addition of the PP7 domain or the TAR domain significantly improve PE- VLP performance as compared with addition of the MS2 stem loop domain.

[0405] Improved pegRNA and GAG PE versions were then combined to estimate the performances of PE-VLPs equipped with the best options.

[0406] The GAG-PE V2, GAG-PE V3 and GAG-PE V4 constructs were obtained.

[0407] GAG-PE V2 (deltaRNase H) has a DNA sequence of SEQ ID NO: 22 and an amino acid sequence of SEQ ID: 23.

[0408] GAG-PE V3 has a DNA sequence of SEQ ID NO: 24 and an amino acid sequence of SEQ ID NO: 25.

[0409] GAG-PE V4 has a DNA sequence of SEQ ID NO: 27 and an amino acid sequence of SEQ ID NO: 28.

[0410] The following ePegRNAs were also obtained: ePegRNA YFP (SEQ ID NO: 27), PegRNA YFPs TAR-trimmedEvoPreQi (SEQ ID NO: 29), PegRNAYFPs MS2-trimmedEvoPreQi (SEQ ID NO: 30), PegRNA YFPs PP7-tnmmedEvoPreQi (SEQ ID NO: 31)

[0411] 4x106HEK293T cells were seeded 24h prior to transfection with a GAG-PE, one or more PegRNA and a mix of envelope proteins (VSVG / BaEV / Syncitine). In proportion, around 16 % of GAG-PE, 37% of GAG-POL, 19% of envelopes and 28% of guide RNA were obtained. 48h after transfection the supernatant was harvested, clarified by centrifugation (400g, 10 minutes) and concentrated by ultracentrifugation (lh!5, 7°C and 35605g). Subsequently, pellets were resuspended in a volume corresponding to 1 / 100thof thesupematant volume. For a lOmL supernatant volume, pellets were resuspended in lOOpL filtered PBS IX. For transduction assay, 1x104recipient HEK293T cells expressing a monocopy YFPs cassette were seeded in a 96 well plate. The next day, 5pL of VLPs were inoculated to transduce them. 72-96 hours posttransduction cells were prepared for FACS analysis.

[0412] Editing efficiencies of HEK293T transduced by YFPs-VLPs were measured. Three independent batches of VLPs were prepared for each condition and used to transduced single-copy YFPs reporter cells. Control productions performed without GAG-PE and non transduced condition (NTD) were added leading to undetectable editing. Fold change relative to epegRNA VI.2 normalized on the VLPs PE VI was provided (this corresponds to the VLP comprising GAG-PE VI and the pegRNA including MS2-trimmedEvoPreQl). Taken together, these improvements of the pegRNA design and the Prime Editor boosted by close to 4 to 9-fold editing efficiencies in HEK293T reporter cells as compared with the original prime editor system (Figure 6A). This effect was confirmed on other cell types including the A549- YFPs reporter cell line and the U2OS-YFPs reporter cell line where the editing efficiencies were boosted by 15 fold (Figure 6B).

[0413] Example 5: Capture-proteins embedded within VLPs ensure active packaging of epegRNA

[0414] Having inserted the MS2 stem loop within a epegRNA comprising a trimmedEvoPreQi, the inventors reasoned that this particular motif may allow the active packaging of epegRNAs within nascent particles. To test this hypothesis, the inventors constructed a GAG-MCP having a DNA sequence of SEQ ID NO: 32 and an amino acid sequence of SEQ ID NO: 33) and produced VLPs with optimal amounts of GAG-MCP added into producer cells. The inventors investigated different proportions of GAG and GAG MCP in the DNA mixture leading to production of particles after transfection. Total GAG construct DNA was fixed at 100%. Editing efficiencies of VLPs produced with all recipes were analyzed by FACS. The inventors found that GAG MCP should be added at low concentration and its propotion relative to other GAG constructs should not exceed 12 % to ensure an optimal product! on, this represents a maximum of 5.4% of the total amount of DNA used for transfection.

[0415] To check whether this system could capture additional epegRNAs molecules within VLPs, the inventors purified total RNA from concentrated particles and quantified the epegRNA by RT- qPCR. They found that the quantity of epegRNA detected by this approach was increased when the GAG-MCP protein was added into the VLP recipes and only if the epeRNA was equipped with the MS2-stem loop.

[0416] Encouraged by these findings, the inventors replaced the MS2 stem loop by other RNA domains known to interact with RBPs with high affinity. The inventors investigated the PP7CP / PP7 tandem and the TAT / TAR tandem by constructing epegRNA modified to incorporate the TAR domain or the PP7domains. They also fused GAGmlv to two versions of hivTAT and to PP7CP and tested whether VLPs incorporating those GAG-RBP could package efficiently an epegRNA equipped with the appropriate RNA domain. The sequences of the corresponding pegRNAs are provided as SEQ ID NO: 31 (PegRNA YFPs PP7-trimmedEvoPreQi) and SEQ ID NO: 29 (PegRNA YFPs TAR-trimmedEvoPreQi). The GAG-PP7CP construct has a DNA sequence of SEQ ID NO: 34 and an amino acid sequence of SEQ ID NO: 35.

[0417] The GAG-TAT1 construct has a DNA sequence of SEQ ID NO: 36 and an amino acid sequence of SEQ ID NO: 37.

[0418] The GAG-TATldelta2-26 construct has a DNA sequence of SEQ ID NO: 38 and an amino acid sequence of SEQ ID NO: 39. qPCR analysis of VLP-RNAs revealed that MCP, PP7CP and TAT containing VLPs could capture epegRNAs modified with the cognate RNA structure (Figure 7).

[0419] Example 6: Versatility of PE- VLPs

[0420] Besides the above-mentioned epegRNA inserting a nucleotide triplet within the HEK3 locus, the inventors attempted to package a pegRNA that could impose a deletion, notably a 5 nucleotide deletion in the same HEK3 locus.

[0421] Five VLPs batches were prepared for this pegRNA leading to editing efficiencies close to 20% for the best batch.

[0422] The following pegRNAs were obtained: PegRNA TAR-trimmedEvoPreQi HEK CTT insertion (SEQ ID NO: 40), PegRNA TAR-trimmedEvoPreQi HEK3 l-5deletion (SEQ ID NO: 41) and PegRNA TAR-trimmedEvoPreQi RNF2 GTA insertion (SEQ ID NO: 42).

[0423] 4x106HEK293T cells were seeded 24h prior transfection with GAG-PE V4, one or more aforementioned PegRNA and a mix of envelope proteins (VSVG / BaEV / Syncitine), the ratios used are the same as those described in example 4. 48h after transfection, the supernatant was harvested, clarified by centrifugation (400g for 10 minutes) and concentrated by ultracentrifugation (Ihl 5, 7°C and 35605g). Subsequently, pellets were resuspended in a volume corresponding to 1 / 100thof the supernatant of the volume. For a lOmL supernatant volume, pellets were resuspended in lOOpL filtered PBS IX. For transduction assay, 1x104recipient HEK293T cells were seeded in a 96 well plate. The next day, lOpL of VLPs were inoculated to transduce them. 96 hours post-transduction cells were harvested and genomic DNA was extracted with the Nucleospin DNA RapidLyse Kit by following manufacturer instructions (Macherey Nagel, 740100.250). The targeted locus was PCR amplified and the level of prime editing was determined by sanger sequencing and TIDE analysis. In addition to the cell lines mentioned above, the inventors experimented PE-VLPs on the HAP-1 cell line and the K562 cell line, a chronic myelogenous leukemia cell line which serves as a model for human haematopoiesis. CTT insertion was measured by TIDE in HAP-1 cells and K562 cells after transduction with PE-VLPs editing HEK3. Editing efficiencies achieved by PE-VLPs in these cells are given in Figure 8 and reached 9% for HAP-1 and 6% for K562. The transfected K562 target cells were prepared in the same manner as above, by seeding 2x104cells in a 96 U- bottom plate.

[0424] More interestingly HEK3 PE-VLPs were also tested on human induced pluripotent stem cells (hiPSC).

[0425] PE VLPs produced from HEK293T were prepared using the described plasmid mixture and 3% of VSV-G encoding plasmid and 7% of BRL encoding plasmid. The day after transfection medium was replaced by Optimem supplemented with penicillin / streptomycin (5%) and Glutamax. VLPs containing medium (15ml) was harvested 48h post transfection, clarified by centrifugation (3 min at 700g) and centrifuged 45min in a Vivaspin 20 concentration device (50.000 MWCO). Resulting concentrated particles (around 200 ul) were next used directly on iPS cells plated in a 12-w plate in 400ul of final volume. VLPs were administrated in one inputs or two inputs (*) separated by 24h. 2h after each VLP administration, transduction medium was supplemented with 1ml of fresh iPS medium. 72h after the last transduction, cells were frozen and genomic DNA extracted before amplification of the HEK3 locus and sanger sequencing.

[0426] Results were given as a TIDE analysis giving the frequency of insertion and deletion in a sequenced locus. Insertion of the triplet increased with dose of VLPs as given in the dose response curve.

[0427] CTT insertion in HEK3 measured by Sanger sequencing / TIDE analysis indicated that VLPs edited these highly valuable cells with an efficiency of 6,9% without apparent toxicity. CTT insertion was further confirmed by a PCR assay detecting the CTT insertion. This level was comparable to efficiencies measured in iPSc by other groups using more invasive transfection techniques, such as described in Li et al. (Li, H. et al. Highly efficient generation of isogenic pluripotent stem cell models using prime editing. (2022): https: / / doi.org / 10.7554 / eLife.79208). 6, A, pegRNA multiplexing

[0428] Moreover, the inventors explored the possibility to package several pegRNA within the same VLP preparation, an approach that could allow multiple edits in the same cell and could increase the percentage of editing by exploiting selection systems.

[0429] This was achieved by preparing VLPs (GAG-PE V4 + ePegRNA VI.2 as described elsewhere) targeting both the HEK3 locus and the RNF2 locus. For this pegRNAs targeting, the HEK3 locus (epegRNAvl.2) and the RNF2 locus (epgRNAvl.2) were mixed in the VLP recipe. After transduction with increasing amounts of concentrated VLP, genomic DNA from treated cells were extracted and genotyped by Sanger sequencing and TIDE analysis. Percentages of CTT insertion in the HEK3 locus and GTA insertion in the RNF2 locus are measured.

[0430] Results given in Figure 9 show that PE- VLPs can accommodate at least two different pegRNAs and ensure their co delivery. This observation indicate that PE- VLPs can also codeliver helper gRNAs that are associated with a pegRNA to optimize editing in the PE3 system.

[0431] 6, B, Protein copackaging

[0432] Finally, the inventors investigated whether VLPs could also package additional proteins that may exert their action in target cells in addition with the prime-editing RNPs (ribonucleoproteins).

[0433] To verify this notion, VLPs were produced with the adjunction of a GAG-mCherry construct supplementing the classical recipe of PE- VLP preparation.

[0434] PE VLPs packaging the YFPs pegRNA were produced with or without the addition of GAG- mCherry within producer cells. For the production of such VLPs, mCherry encoding plasmids were added in the transfection mix. The proportion of said plasmids was 5% of the total DNA transfection. Once expressed, the GAG-mCherry can be incorporated within VLPs rendering them fluorescent (detectable by Virometry) and able to transmit this features to transduced cells. Fluorescent-protein delivery was thus concomitant with delivery of PE-RNPs allowing a trackability of transduction. To verify that this incorporation of labelling protein within particles did not affect their efficiency in delivering PE, PE- VLPs with or without mCherry were produced in parallel (3n each) and compared for their capacity to convert YFPstop to YFP in the HEK293T reporter cell line.

[0435] It was found that PE- VLPs labelled with GAG-mCherry were still able to deliver PE in a reporter cell line without significant loss of efficacy. Editing efficiencies mean was close to 15% for non- labelled PE-VLPs and 13% for labelled PE-VLPs indicating that GAG-mCherry incorporation did no impair the assembly of PE-VLPs significantly nor their ability to deliver the PE-RNPs.

[0436] This may allow the characterization of particle preparations (Virometry analysis) or the sorting of transduced cells.

[0437] More generally this capacity of PE-VLP to host proteins beyond PE components may allow the incorporation of helping factors known to potentialize prime editing.

[0438] Collectively these data show that PE-VLPs according to the invention can deliver PE into five different human cell-lines, into human iPSc, can package different pegRNAs alone or in combination and can host other proteins of interest.

[0439] Example 7: Fidelity of editing mediated by PE VLPs

[0440] Since this study combined PE with a RNP-delivering system, the inventors wanted to investigate how the delivery of PE components by VLPs could further enhance the accuracy of the editing process.

[0441] To address this question, they designed mismatch-tolerance assays mimicking an imperfect hybridization of the epegRNA with its target sequence. In the first assay, mutations were added in the target sequence and for the second assay, mutations were added on the HEK3 epegRNA. Using these two systems, they measured fidelity of prime editing delivered by plasmid transfection - the technique employed in a vast majority of studies- or by PE-VLPs.

[0442] Fidelity assay YFPs: mutating positions in the target

[0443] For their first fidelity assay, the inventors introduced one or two mismatches into the YFPs cassette.

[0444] Cassette SWYM1MM contained one mismatch and had the DNA sequence of SEQ ID NO: 43, from ATGto the final stop.

[0445] Cassette SWYM2MM contained two mismatches and had the DNA sequence of SEQ ID NO: 44, from ATGto the final stop.

[0446] These modifications of the target sequence compromise the hybridization of the epegRNA both at the level of the crRNA and the PBS. If properly repaired, these mutant cassettes should express a fully fluorescent protein since imposed mismatched did not modify the amino acids of YFP.

[0447] These cassettes were next transduced into HEK293T reporter cells by saturating lentiviral transduction (moi40) to increase the number of copies into recipient cells and facilitate the measurement of low frequency editing-events. The transduced HEK293T cells were used as targets to measure editing efficiencies of Prime Editing delivered with plasmid transfection or PE- VLP transduction.

[0448] Transfection with PE / pegRNA was done in the same cell density conditions as in previous examples. Results were analysed 96 hours after transfection / transduction, to ensure both techniques (transfection / transduction) reached their optimal action.

[0449] Three cell lines harbouring no mutation within the YFPs cassette, one mismatch or two mismatches were edited by plasmid transfection or by two independent batches of PE-VLPs (Figure 10).

[0450] Cell editing by transfection (orange bars) or two preparations of PE-VLPs was analysed by FACS. Editing efficiencies of a perfect YFPs cassette (left panel) were close to 70% for transfection and 50% for transduction with PE-VLPs. Interestingly, the inventors still measured significant editing signals into transfected cells bearing one mismatch at the level of the YFPs target (close to 5% medium panel) and two mismatches (0,25% right panel).

[0451] On the other hand, VLPs transduction led to very low editing in mismatched reporter cells.

[0452] Depending on the experiment, the inventors noted that PE-VLPs transduction was systematically more precise than transfection in this assay by a factor ranging from 36 to 377-fold for one mismatch and 3 to 28-fold for two mismatches into the YFPs target sequence.

[0453] Fidelity assay HEK3: mutating positions in the epegRNA

[0454] The inventors next followed a similar approach by introducing single mismatch in the epegRNA that mediates the insertion of a CTT-triplet within the HEK3 endogenous locus. The corresponding ePegRNA has the sequence of SEQ ID NO: 45.

[0455] Different positions were mutated in the crRNA and in the PBS region as detailed below. Sequences are given as the pegRNA DNA plasmids, from the first nucleotides of the pegRNA to the end of the pegRNA (tttt). The following mutants were prepared: t!7 (SEQ ID NO: 46), t!4 (SEQ ID NO: 47), tlO (SEQ ID NO: 48), g6 (SEQ ID NO: 49), t3 (SEQ ID NO: 50), pAl (SEQ ID NO: 51), pA5 (SEQ ID NO: 52), pG9 (SEQ ID NO: 53).

[0456] All constructs were next used to edit the HEK3 locus by transfection (they were co-transfected with a CAS9n-RT construct) or by PE-VLPs transduction. CTT insertions in HEK3 were next monitored in treated cells by systematic sequencing of the HEK3 locus and TIDE analysis. Sanger sequencing the HEK3 locus revealed how mutations in the pegRNA affected the efficacy of editing, delivered either by DNA transfection or by PE-VLP transduction.

[0457] Results are given in Figure 11 as the relative editing efficiencies of two independent experiments. Editing efficiencies measured for non-mismatched pegRNA were set at 100% for both transfection and transduction assays.

[0458] Interestingly, it was noted that mutations close to the PAM were less permissive than mutations in the 3’ part of the pegRNA as it has been already described for this particular epegRNA and more generally for gRNA in the Cas9 classic CRISPR system. For example, imposing one mismatch in the middle of the crRNA region (T10 mutant) appeared to be well tolerated since 60% of editingactivity remained detected in the transfection condition, while this value decreased to less than 20% when the mismatch was closer to the PAM (mutant T17). By comparing two independent transfection experiments with two independent transduction assays, the inventors found that PE- VLPs were more accurate than transfection with a factor ranging from 1,5 to 70-fold depending on the mismatched position in the crRNA.

[0459] In conclusion, the data provided above shows that VLPs according to the invention are able to deliver Prime editing in human cells, including iPSc.

[0460] The inventors were able to demonstrate that the molecular improvements of constructs of the Prime editing tool enabled enhanced fidelity of editing, in particular compared to plasmid transfection.

[0461] Example 8: Generation of PE- VLPs that incorporate an optimized Prime editor architecture The inventors next attempted to replace the original Cas9n-RT (PE) by the PEmax version and evaluated the efficacy of VLPs of the invention loaded with a epegRNA correcting the stop codon ofYFP.

[0462] PEmax is an optimized sequence of the Cas9n-RT fusion that surpasses the original PE2 system to edit Hela cells. It results from a tedious large-scale test of several constructs harboring mutations within Cas9n-RT, engineered linkers, and codon-optimization (see Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635- 5652. e29 (2021)).

[0463] This design was poorly efficient to transfer Prime editing (compare VI with Vim).

[0464] Thus, the inventors explored different designs of GAG-PEmax by testing different linkers including or not the Nuclear export signal of HIV (NES) and modifying their positions within the construct. V3-V4 exploit the original CAS9n while Vlmp, V2mp and V3m / mp-V4m / mp exploit the optimized CAS9n-RT max (PEmax). All V2m, V4m and V4 mp versions were deleted for the RNase H domain within the RT. Vlmp-V4mp constructs incorporate a linker (SP-3NES) in Cterminus of the fusion protein rather than in between the GAG and prime editor. A protease site is included between GAG and the prime editor. NES stands for the nuclear export signal of EHV- 1.

[0465] Table n°2

[0466] These explorations were performed on the GAG-Pemax construct containing a complete RT domain (RT or Rtmax) or a RNase-H deleted domain (RT ARNaseH or Rtmax ARNaseH).

[0467] Figure 12 provides the structural details of all the fusion constructs generated and their name.

[0468] All constructs were next used to produce VLPs and compared for their efficacy to deliver PE into YFPs-reporter cells. As shown in Figure 13, the inventors noted major differences between all constructs tested and pointed out the higher performance of the Vlmp construct.

[0469] Insertion of the NES linker in C-terminus of the “max” version of GAG-Cas9n-RT fusion instead of between GAG and Cas9n-RT seems highly beneficial to the production of Pemax-VLP. Except for V4mp, the inventors found that the presence of two NES linkers within the same editor seem detrimental to production of efficient VLPs.

[0470] This assay allowed to identify an optimal GAG construct fused to Pemax (Vlmp) which appears to be 1,5-fold more efficient that the previously described designs.

[0471] The corresponding constructs were obtained: GAG-Pemax (Vim), GAG-PEmaxARNaseH (V2m), GAG-3NES-Pro-Pemax (V3m), GAG-3NES-Pro-PEmaxARNaseH (V4m), GAG-Pemax- Pro-3NES (Vlmp), GAG-PemaxARnaseH-Pro-3NES (V2mp), GAG-3NES-Pro-Pemax-Pro- 3NES (V3mp) and GAG-3NES-Pro-PemaxARnaseH-Pro-3NES (V4mp).

[0472] GAG-Pemax (Vim) has the DNA sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 55.

[0473] GAG-PEmaxARNaseH (V2m) has the DNA sequence of SEQ ID NO: 56 and the amino acid sequence of SEQ ID NO: 57.

[0474] GAG-3NES-Pro-Pemax (V3m) has the DNA sequence of SEQ ID NO: 58 and the amino acid sequence of SEQ ID NO: 59.

[0475] GAG-3NES-Pro-PEmaxARNaseH (V4m) has the DNA sequence of SEQ ID NO: 60 and the amino acid sequence of SEQ ID NO: 61.

[0476] GAG-Pemax-Pro-3NES (Vlmp) has the DNA sequence of SEQ ID NO: 62 and the amino acid sequence of SEQ ID NO: 63.

[0477] GAG-PemaxARnaseH-Pro-3NES (V2mp) has the DNA sequence of SEQ ID NO: 64 and the amino acid sequence of SEQ ID NO: 65.

[0478] GAG-3NES-Pro-Pemax-Pro-3NES (V3mp) has the DNA sequence of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 67.

[0479] GAG-3NES-Pro-PemaxARnaseH-Pro-3NES (V4mp) has the DNA sequence of SEQ ID NO: 68 and the amino acid sequence of SEQ ID NO: 69.

[0480] Example 9: Inducible expression of the epegRNA by a strong hCMV promoter

[0481] To increase their toolbox and extend the versatility of PE- VLPs -notably to package T-rich pegRNAs-, the inventors generated constructs that encode an YFPs pegRNA within the intronic sequence of a luciferase-coding plasmid (Figure 14). The pegRNA sequence was inserted within the intronic sequence of a Firefly-Luciferase encoding plasmid and surrounded by two Csy4 stemloops as depicted. Processing of the intron and release of the pegRNA was ensured by the Csy4 protein that cleaves the 3’ of the csy4 stem-loop(sl). Thus the production of a pegRNA available for pairing with CAS9n was dependent on the expression of the Csy4 protein. Indeed, upon expression of the Csy4 protein, the pegRNA was cleaved and released from the intron while the processed RNAs expressed the Luciferase mRNA.

[0482] To clarify whether this system could be beneficial to VLPs efficacy, three different pegRNAs carrying diverse stabilizing structures were engineered:

[0483] Table n°3

[0484] Resulting PE- VLPs were compared for their capacity to repair the YFPs once delivered onto reporter cells. In this approach, VLPs were produced after the transfection of a plasmid mixture including a plasmid encoding the Csy4 protein, or its mutant Csy4H29A devoid of cleaving activity. The proportion of plasmids encoding the Csy4 protein or its mutant was 5% of the total DNA transfection. Editing was analysed by FACS as the % of YFP correction 3 days after transduction. For each transduction assay, three VLP batches were produced independently. Comparisons of efficiencies were calculated and compared with pegRNAS obtained via the V2 guide system (POL III driven).

[0485] Results given Figure 15 show that VLPs resulting from this particular expression system are highly efficient in editing reporter cells and even surpasses the classical U6-driven system (epegRNAV2). Interestingly, expression of the H29A mutants fails to produce efficient VLPs while the inventors noted that luciferase loaded particles were indeed produced (results not shown). This indicates that expression of the pegRNA is highly dependent on the processing of the intron by Csy4.

[0486] Furthermore, the inventors noted that the csy4-stem loop structure which remains in the C- Terminus extremity of the pegRNA after cleavage may serve as a stabilization structure as efficient as the TAR-tEvoPreQi tandem previously described. Placing a TAR sequence between the pegRNA PBS and the csy4-stem loop domains further increases the efficacy of VLPs (pegRNA-TAR-Csy4) while insertion of an additional trimmedEvoPreQi domain does not seem to further potentialize the global efficacy of our VLP system (ePegRNAV2-Csy4). Altogether these data show that expression of pegRNA by the POL II CMV promoter boosts VLPs production of efficient PE-VLPs, and that this expression is inducible by the expression of Csy4 protein. Thus Csy4-VLP production is limited to cells expressing transiently Csy4 or engineered to stably express it. More importantly this system allows to produce high loads of VLPs packaging any pegRNA, including those that incorporate a T-rich stretch. Thanks to the use of the FLUC plasmid, those Csy4-VLPs package a passive cargo of FLUC protein that can be easily dosed upon lysis of VLPs. Moreover, the inventors have shown that addition of the two domains (Csy4-stemloop plus TAR) in the 3’ extremity of the pegRNA surpasses the stability conferred by the previously described TAR-trimmedEvoPreQi domain and that all Pol-II PEgRNAs showed improved YFPs-edition over the Pol-III counterpart.

[0487] Next, the inventors explored if the improvement of the Prime Editor Pemax (Vlmp) described above (Example 8) could synergize with this Csy4-expressing system. To test this, they produced VLPs loaded with YFPs pegRNA (pegRNA-TAR-Csy4) and compared the efficacy of three independent VLPs batches to mediate editing within the YFPs reporter cell line.

[0488] Results indicated that the combination of Vlmp with the Csy4 expressed pegRNA generated VLPs that saturated the reporter cell line (close to 100% of editing for batch 1) and was significantly more efficient to produce VLPs than the V4 system (as described in Example 3) (see Figure 16).

[0489] In this experiment, producing cells were seeded 24h before transfection with 6x105cells in a 6- well plate. Then transfection was carried out with the ratio previously mentioned in example 4. 48h after transfection, supernatants were harvested and clarified by centrifugation (400g during 10 minutes) and then concentrated by centrifugation at 10000g for 6h. Pellets were resuspended in a volume of filtered PBS IX corresponding to l / 40thof the original supernatant volume (50pL for 2000pL of supernatant). 1x104HEK293T reporter cells were seeded 24hours prior to transduction assay. Transduction was carried out with 5pL of concentrated VLP. 72-96h post transduction, cells were prepared for FACS analysis.

[0490] Finally, the inventors designed pegRNAs to insert three nucleotides within the HEK3 locus or the RNF2 locus. Two designs were evaluated carrying either the TAR-tEvoPreQl structures in 3’ or the TAR structure associated with the csy4 stem loop that remains on the RNA after intron cleavage. The pegRNAs are as follow: PegRNA TAR-Csy4 HEK3 CTT insertion (SEQ ID NO: 74) and PegRNA TAR-Csy4 RNF2 GTA insertion (SEQ ID NO: 75).

[0491] Respectively, these RNAs were encoded by a U6 promoter or a CMV promoter. Two different editors were evaluated: a GAG-PE fusion protein where GAG and PE are separated by a SP- 3NES-SP linker (V4 and V1.2mp), and a GAG-Pemax fusion protein, wherein a SP-3NES linker is inserted in C-terminus of the protein (V4C and VlmpC).

[0492] 10 000 cells were transduced by 5 pl of 3 different batches of concentrated VLPs (concentrated using 10 000 RCF (relative centrifuge force)). Results are provided in Figure 17 as the percentages of triplet insertion as measured by TIDE after genome sequencing on the HEK3 locus or the RNF2 locus.

[0493] These show that for an “easy-to-modify” locus (HEK3 locus), all four combinations were efficient to edit the locus. However, when the inventors targeted a difficult-to-edit loci like RNF2, the VlmpC (pegRNA encoded by a CMV promoter and GAG-PE fusion protein wherein a SP-3NES linker is in C-ter) system improve editing efficacy by 2.7-fold (compare 2.57% Vs. 6.87%).

[0494] Example 10: Multiplexing of pegRNA; VLPs loaded with several pegRNA may allow selection of edited cells

[0495] The inventors attempted next to produce VLPs loaded with equimolar amounts of a epegRNA targeting YFPs and a epegRNA targeting the HEK3 locus (the chosen design for both of them was TAR-tEvoPreQi). Resulting PE- VLPs were used to prime-edit YFPstop cells which expressed YFP three days after PE -VLPs transduction.

[0496] The inventors next sorted YFPs positive cells and questioned whether these cells edited thanks to the first epegRNA(YFPs) could be also edited by the second HEK3 epegRNA.

[0497] Results provided in table 4 give the editing enrichment of edited HEK3 locus in the sorted and non-sorted population. These HEK3 -editing values were obtained after cell lysis of sorted cells and sanger sequencing of the HEK3 targeted locus prior to TIDE analysis.

[0498] Table 4 The inventors noted that YFPs positive cells were significantly enriched with cells edited for the HEK3 locus (1,3-1, 5 fold). This was observed for several VLPs batches and several transduction assays. This observation indicates that PE- VLPs can be engineered to package at least two pegRNAs. Should this multiplexing option include a pegRNA permitting a selection of edited cells, -imposing for example a drug-dependence, a surface marker alteration or the expression of an internal staining as performed here-, this may allow to set up strategies to enrich transduced populations with edited cells.

[0499] Example 11: Generation of PE- VLPs that incorporate a PE6 prime editor

[0500] The inventors next attempted to replace the PEmax prime editor from the VLPs described in Example 8 above by PE6 prime editors and evaluated the efficacy of VLPs of the invention loaded with pegRNAs inserting either a 3nt CTT, a FLAG sequence or two 25nt sequences and comprising TAR and Csy4 structures in 3’.

[0501] For the production of the VLP, the method remained similar to that described for GAG-PEmax VLPs but using a production method where the pegRNA sequence was placed within the intron of a Pol II driven construct (see Example 9). The pegRNA sequence was released from the intron in produced cells by the action of the Csy4 nuclease.

[0502] Briefly, constructs encoding the viral proteins GAGPOL, the envelopes VSVG, BRL, and Syncytin were co-transfected into HEK293T cells in the presence of Csy4 (Cas6f nuclease), editors, and pegRNA. 48h after transfection, the inventors harvested the supernatant and purified the VLPs. They then transduced HEK293T cells plated 24h prior. 72h after transduction, cells were harvested and their DNA extracted. Following, the HEK3 locus were PCR amplified and the editing percentage determined by sanger sequencing and tide analysis.

[0503] The following pegRNA sequences were used for inserting either a 3nt CTT (SEQ ID NO: 74), a FLAG sequence (SEQ ID NO: 101) or a pinned 25nt sequence (SEQ ID NO: 102) and an unpinned 25 nt sequence (SEQ ID NO; 103).

[0504] The inventors were able to explore different designs of GAG-PE6 by testing different linkers including or not the Nuclear Export Signal of HIV (NES) and modifying their positions within the construct. The constructs were created by fusing the GAG protein from Murine Leukemia Virus (MLV) with various editors PE6b, PE6c and PE6d as described in Doman et al., Cell 2023. The PE-VLP constructs were modified by inserting a proteolytic site (sp) at the GAG-PE junction, or a cleavable linker of the invention comprising proteolytic sites (sp) and nuclear export signals (NES) at different positions: at the GAG-PE junction, at the C-terminus, or at both positions.

[0505] The constructs are as follows: The GPE6b construct contains a proteolytic site between GAG and PE6b, but no cleavable linker according to the invention. The G3nPPE6b construct contains a cleavable linker of the invention at the GAG-PE6b junction, said linker comprising, from N-ter to C-ter, a first proteolytic site, three NES, and a second proteolytic site. The GPE6bP3n construct contains a cleavable linker of the invention in C-ter, said linker comprising, from N-ter to C-ter, a proteolytic site, and three NES. The G3nPPE6bP3n construct contains a first cleavable linker according to the invention between GAG and PE6b, and a second cleavable linker of the invention in C-ter. Similar constructions have been made with PE6c, namely GPE6c, G3nPPE6c, GPE6cP3n and G3nPPE6cP3n, respectively. Similar constructions have been made with PE6d, namely GPE6d, G3nPPE6d, GPE6dP3n and G3nPPE6dP3n, respectively (see Table 5). Figure 18 provides the structural details of all the fusion constructs generated and their name.

[0506]

[0507] Table 5

[0508] The editing percentage for each construct was measured by Sanger sequencing of a purified PCR product, obtained after PCR amplification of the HEK3 locus on the edited cells.

[0509] As shown in Figures 18 B-E, the inventors noted major differences between all constructs tested: PE-VLP constructs encompassing PE6b were designed to insert the triplet CTT and the FLAG sequence (24 nts) into the HEK3 locus as shown in figures 18 B and C, respectively. While all constructs allow efficient editing, PE6b demonstrated superior performance in VLP-mediated genome editing compared to VLPs with PEmax, with higher editing efficiencies for both CTT and FLAG insertions in HEK293T cells. Moreover, no indel events were detected with PE6b.

[0510] PE-VLP constructs encompassing PE6c and PE6d were designed to insert structured (pinned) or non-structured (unpinned) sequences. In figures 18 D and E, construct GPmP3n with PEmax showed high editing efficiency, particularly for structured (pinned) inserts.

[0511] Regarding the positioning of the linkers in the PE6-VLP constructs, insertion of the PS-3xNES motif at the C-terminus of the editor consistently improved VLP efficiency compared to insertion at the GAG-PE junction. The fold-increases in efficiency between the two designs are illustrated in Figure 18 B-E. Specifically, the efficiency increases are as follows: a 1.34-fold increase for CTT insertions using construct GPR6bP3n as compared to G3nPPE6b, a 1.44-fold increase for FLAG insertions using construct GPR6bP3n as compared to G3nPPE6b, a 1.57-fold increase for pinned RTT insertions using construct GPR6dP3n as compared to G3nPPE6d, and a 1.92-fold increase for unpinned RTT insertions using construct GPR6cP3n as compared to G3nPPE6c. However, double introduction of 3xNES at both the C-terminus and the GAG-PE junction was generally less efficient than a single insertion at the C-terminus for all tested editors.

[0512] Together, these findings highlight the potential of PE6 prime editors for VLP-mediated genome editing. PE6b is a highly efficient editor for short, simple genome editing, surpassing the performance of the PEmax system for CTT and FLAG sequences. PE6c and PE6d are tailored for more complex edits, including long insertions or edits requiring highly structured reverse transcription templates. Besides, the strategic positioning of the sp-3xNES domain at the C- terminus of the editor significantly enhances VLP efficiency, providing a valuable insight for optimizing future genome editing tools. LIST OF SEQUENCES

[0513] SEQ ID NO: 1: Amino acid sequence of VSVG

[0514] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQV KMPKSHQAIQADGWMCHASKWVTTCDFRVYGPKYITHSIRSFTPSVEQCKESIEQTKQG TWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTV HNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACK MQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRM VGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKA QVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVL RVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0515] SEQ ID NO: 2: DNA sequence of VSVG atgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgt tccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcac caggctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgcgtgtatggaccgaagtatataacacattcc atccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaa agttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgg gttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtcaaagg gctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcaga agtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcg agatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgta agtctaattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtgg atctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagag tcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgggcaccatatg aagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccgatc ttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactg ggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggac tattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttgg aaagtaa

[0516] SEQ ID NO: 3: Amino acid sequence of BaEV-R-Less (BRL)

[0517] MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSG KTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTA TLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQ RI<LEEIHI<ALYPELQYHPLAIPI<VRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLC LKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVA FSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIP AIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDL QDQVDSLAEWLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERR RKDLASNPLWTGLQGLLPYLLPFLGPLLTLLLLLTIGPCIFNRLTAFINDKLNIIHAM

[0518] SEQ ID NO: 4: DNA sequence of BaEV-R-Less (BRL) atgggattcacaacaaagataatcttcttatacaacctagtactggtctacgcggggtttgacgaccctcgcaaagccatagaactagtacaaa agcgatatggccgaccatgcgattgcagcggaggacaagtgtccgagcccccgtcagacagggtcagtcaagtgacttgctcaggcaaga cagcttacttaatgcccgaccaaagatggaaatgtaagtcaattccaaaagacacctccccaagcgggccactccaagagtgcccctgtaatt cttaccagtcctcagtacacagttcttgttatacctcataccaacaatgcagatcaggcaataagacatattatacggctactctgctaaaaacac aaactgggggcaccagtgatgtacaagtattaggatccaccaacaaacttatacaatctccctgtaatggcataaaagggcagtctatttgctg gagcactacagctcctatccacgtctctgatggaggaggtccattagacaccacaagaattaaaagtgttcagagaaaactggaagaaattca taaagccctatatcctgaacttcagtatcaccctttggccatacctaaggttagagataacctcatggtcgatgcccagactttaaacattctcaat gccacttacaacttactcctaatgtccaacacgagcctagtggacgactgttggctttgtttaaaattaggtccccctactcccctcgcaataccta acttcctattatcctacgtgactcgctcctcggataatatctcttgtttaataattcccccccttctagttcaaccgatgcagttttccaattcatcttgc ctcttttccccctcctacaacagtacagaagaaatagatctaggccatgttgccttcagcaactgtacctccataaccaatgtcaccggtcccata tgcgctgtaaatggttcggtctttctctgtggcaataacatggcatacacttatctacccacgaactggacggggctttgcgtcctagcaactctc ctccccgacattgacatcattcccggagatgaaccggtccccatccctgctattgatcattttatatatagacctaaacgggccatacagtttattc ctttactagcagggctagggatcaccgcagccttcacaacaggagctacaggcctaggtgtctctgtgacccaatatacaaaattatctaatca gctaatttctgatgtacaaatcttatctagcaccatacaagatctgcaagatcaagtagactcattagccgaagtggttctccagaacagaaggg ggctagatctacttacagcagaacaaggaggaatctgtttagccctgcaagaaaaatgctgcttttatgttaacaagtcagggattgtgagaga caaaataaaaaccttacaagaagaactagaaagacgtagaaaagatctagcttccaacccactttggactgggcttcaagggctcctccctta cctcctgccctttcttggccctctacttaccctcctgctcttactcaccattgggccgtgcatttttaaccgtctaaccgcttttattaatgataagttaa acataatacacgctatgtag

[0519] SEQ ID NO: 5: Amino acid sequence of hSyncytin-1

[0520] MALPYEOFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLSKGTPTF TAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDGGG VQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLFNTTLTGLHEVS AQNPTNCWICLPLNFRPYVSIPVPEQWNNFSTEINTTSVLVGPLVSNLEITHTSNLTCVKFS NTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCLNGSESMCFLSFLVPPMTIYTEQD LYSYVISKPRNKRVPILPFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMERVADSL VTLQDQLNSLAAVVLQNRRALDLLTAERGGTCLFLGEECCYYVNQSGIVTEKVKEIRDRI QRRAEELRNTGPWGLLSQWMPWILPFLGPLAAIILLLLFGPCIFNLLVNFVSSR

[0521] SEQ ID NO: 6: DNA sequence of hSyncytin-1 atggccctcccttatcatatttttctctttactgttcttttaccctctttcactctcactgcaccccctccatgccgctgtatgaccagtagctcccctta ccaagagtttctatggagaatgcagcgtcccggaaatattgatgccccatcgtataggagtctttctaagggaacccccaccttcactgcccac acccatatgccccgcaactgctatcactctgccactctttgcatgcatgcaaatactcattattggacaggaaaaatgattaatcctagttgtcctg gaggacttggagtcactgtctgttggacttacttcacccaaactggtatgtctgatgggggtggagttcaagatcaggcaagagaaaaacatgt aaaagaagtaatctcccaactcacccgggtacatggcacctctagcccctacaaaggactagatctctcaaaactacatgaaaccctccgtac ccatactcgcctggtaagcctatttaataccaccctcactgggctccatgaggtctcggcccaaaaccctactaactgttggatatgcctccccc tgaacttcaggccatatgtttcaatccctgtacctgaacaatggaacaacttcagcacagaaataaacaccacttccgttttagtaggacctcttgt ttccaatctggaaataacccatacctcaaacctcacctgtgtaaaatttagcaatactacatacacaaccaactcccaatgcatcaggtgggtaa ctcctcccacacaaatagtctgcctaccctcaggaatattttttgtctgtggtacctcagcctatcgttgtttgaatggctcttcagaatctatgtgctt cctctcattcttagtgccccctatgaccatctacactgaacaagatttatacagttatgtcatatctaagccccgcaacaaaagagtacccattctt ccttttgttataggagcaggagtgctaggtgcactaggtactggcattggcggtatcacaacctctactcagttctactacaaactatctcaagaa ctaaatggggacatggaacgggtcgccgactccctggtcaccttgcaagatcaacttaactccctagcagcagtagtccttcaaaatcgaaga gctttagacttgctaaccgctgaaagagggggaacctgtttatttttaggggaagaatgctgttattatgttaatcaatccggaatcgtcactgag aaagttaaagaaattcgagatcgaatacaacgtagagcagaggagcttcgaaacactggaccctggggcctcctcagccaatggatgccct ggattctccccttcttaggacctctagcagctataatattgctactcctctttggaccctgtatctttaacctccttgttaactttgtctcttccagatga

[0522] SEQ ID NO: 7: NES amino acid sequence

[0523] LPPLERLTL

[0524] SEQ ID NO: 8: 3xNES amino acid sequence

[0525] LPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTL

[0526] SEQ ID NO: 9: Protease site amino acid sequence

[0527] PRSSLYPALTP

[0528] SEQ ID NO: 10: CLEAVABLE LINKER PS-3xNES-PS

[0529] TRSSLYPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTLARPQTRSSLYPAL TP

[0530] SEQ ID NO: 11: CLEAVABLE LINKER PS-3NES

[0531] TRSSLYPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTL

[0532] SEQ ID NO: 12: Nucleic acid of Cas9 H840A nickase atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacatcg gcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcac agcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaa gatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagact ggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaa gtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatg atcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacct acaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacgg ctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaactt caagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccaga tcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagat caccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagca gctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttc tacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcag cggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcct gaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgc ctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcga gcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgag ctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttc aagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtgg aagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggac attctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgac aaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagt ccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaag aggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaag ggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccag agagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagcc agatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgt ggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaaggacgactccatcgacaaca aggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcg gcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataagg ccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacg acgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaag tgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctgg aaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgcc aagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagaca aacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtg aaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaagga ctgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaag aaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagg gctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctct gccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagg gctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctc caagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccga gaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccag caccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtga ctctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagc ggcggcagcagcaccctaaatatagaagatgagtatcggctacatgagacctcaaaagagccagatgtttctctagggtccacatggctgtct gattttcctcaggcctgggcggaaaccgggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctctacccc cgtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggaccagggaatactggta ccctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctgagagaagtcaa caagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctcccaccgtcccaccagtggtacactgtgcttg atttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgcctttgagtggagagatccagagatgggaatctcagga caattgacctggaccagactcccacagggtttcaaaaacagtcccaccctgtttaatgaggcactgcacagagacctagcagacttccggatc cagcacccagacttgatcctgctacagtacgtggatgacttactgctggccgccacttctgagctagactgccaacaaggtactcgggccctg ttacaaaccctagggaacctcgggtatcgggcctcggccaagaaagcccaaatttgccagaaacaggtcaagtatctggggtatcttctaaaa gagggtcagagatggctgactgaggccagaaaagagactgtgatggggcagcctactccgaagacccctcgacaactaagggagttccta gggaaggcaggcttctgtcgcctcttcatccctgggtttgcagaaatggcagcccccctgtaccctctcaccaaaccggggactctgtttaatt ggggcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgccccagccctggggttgccagatttgactaagcccttt gaactctttgtcgacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactgggaccttggcgtcggccggtggcctacctgtcca aaaagctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattgccgtactgacaaaggatgcaggcaagctaac catgggacagccactagtcattctggccccccatgcagtagaggcactagtcaaacaaccccccgaccgctggctttccaacgcccggatg actcactatcaggccttgcttttggacacggaccgggtccagttcggaccggtggtagccctgaacccggctacgctgctcccactgcctgag gaagggctgcaacacaactgccttgatatcctggccgaagcccacggaacccgacccgacctaacggaccagccgctcccagacgccga ccacacctggtacacggatggaagcagtctcttacaagagggacagcgtaaggcgggagctgcggtgaccaccgagaccgaggtaatct gggctaaagccctgccagccgggacatccgctcagcgggctgaactgatagcactcacccaggccctaaagatggcagaaggtaagaag ctaaatgtttatactgatagccgttatgcttttgctactgcccatatccatggagaaatatacagaaggcgtgggtggctcacatcagaaggcaa agagatcaaaaataaagacgagatcttggccctactaaaagccctctttctgcccaaaagacttagcataatccattgtccaggacatcaaaag ggacacagcgccgaggctagaggcaaccggatggctgaccaagcggcccgaaaggcagccatcacagagactccagacacctctaccc tcctcatagaaaattcatcaccctctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaa

[0533] SEQ ID NO: 13: Amino acid sequence of Cas9 H840A nickase

[0534] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE

[0535] NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT

[0536] RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED

[0537] IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE

[0538] NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK

[0539] GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDF

[0540] PQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPC QSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLAD FRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY

[0541] LGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLT KPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVK QPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG

[0542] TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAEL IALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEIL ALLKA LFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRT ADGSEFEPKKKRKV

[0543] SEQ ID NO: 14: DNA sequence of NESI ctgcctccacttgaaagactgacactg SEQ ID NO: 15: DNA sequence of NES2 cttccgcctcttgagagattgacatta

[0544] SEQ ID NO: 16: Amino acid sequence of Cas9n-RT + MLV GAG

[0545] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGGRPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINA SGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN

[0546] FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL

[0547] PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTE

[0548] TEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG

[0549] WLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAIT

[0550] ETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV

[0551] SEQ ID NO: 17: Amino acid sequence of Cas9n-RT + HIV GAG

[0552] MGARASVLSGGELDI<WEI<IRLRPGGI<I<QYI<LI<HIVWASRELERFAVNPGLLETSEGCRQ

[0553] ILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKIEEEQNKSKKKAQQAA

[0554] ADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMFSALSEG

[0555] ATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDI

[0556] AGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVD

[0557] RFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGP

[0558] GHI<ARVLAEAMSQVTNPATIMIQI<GNFRNQRI<TVI<CFNCGI<EGHIAI<NCRAPRI<I<GCW

[0559] KCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQ

[0560] KQEPIDKELYPLASLRSLFGSDPSSQPGTTTPGKARVLAEAMSTGGRPLIRLTIGRAATMK

[0561] RTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK

[0562] KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF

[0563] LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR

[0564] GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI

[0565] AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ

[0566] YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK

[0567] YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD

[0568] NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR

[0569] KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV

[0570] KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRF

[0571] NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM

[0572] KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI

[0573] QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN

[0574] QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ

[0575] ELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQ

[0576] LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE

[0577] NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLE

[0578] SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN

[0579] GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD

[0580] WDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK

[0581] GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL

[0582] KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA

[0583] ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0584] SGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDF

[0585] PQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPC

[0586] QSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL

[0587] DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLAD

[0588] FRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY LGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLT KPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVK QPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAEL IALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEIL ALLKA LFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRT ADGSEFEPKKKRKV

[0589] SEQ ID NO: 18: pegHEK3 ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcgg tcctctgccatcaaagcgtgctcagtctg

[0590] SEQ ID NO: 19: DNA sequence of SWIS cassette agtcgctctgcggagaggctggcagattgagccctgggaggttctctccagcactagcaggtagagcctgggtgttccctgctagactctca ccagcacttggccagtgctgggcagagtggctccacgcttgcttgcttaaagacctcttcaataaagctgccattttagaagtaagccagtgtgt gttcccatctctcctagtcgccgcctggtcaactcggtactcggtaataagaagaccctggtctgttaggaccctttctgctttgagaaaccgaa gcaggaaaatccctagcagattggcgcccgaacaggacttgaaggagagtgagagactcctgagtacggctgagtgaaggcagtaaggg cggcaggaaccaaccacgacggagtgctcctataaaggcgcgggtcggtaccagacggcgtgaggagcgggagaggaggaggcctcc ggttgcaggtaagtgcaacacaaaaaagaaatagctgtcttgttatccaggaagggataataagatagagtgggagatgggcgcgagaaac tccgtcttgtcagggaagaaagcagatgaattgaaaaaattattaatcgcatgaattttaaaagaaggggaggaataggggatatgactccag cagaaagattaattaacatgatcactacagaacaagaaatacaatttcaacaatcaaaaaactcaaaatttaaaaattttcgggtctattacagag ctcacgcgtgattggagttgggagattataaattagtagagatcactccgattggcttggcccccacagatgtgaagaggtacactactggtgg cacctcaagaaataaaagaggggtctttgtgctagggttcttgggttttctcgcaacggcaggttctgcaatgggcgcggcgtcgttgacgctg accgctcagtcccggactttattggctgggatagtgcagcaacagcaacagctgttggacgtggtcaagagacaacaagaattgttgcgact gaccgtctggggaacaaagaacctccagactagggtcactgccatcgagaagtacttaaaggaccaggcgcagctaaatgcttggggatgt gcgtttagacaagtctgccacactactgtaccatggccaaatgcaagtctaacaccagactggaacaatgatacttggcaagagtgggagcg aaaggttgacttcttggaggaaaatataacagccctcctagaagaggcacaaattcaacaagagaagaacatgtatgaattacaaaagttgaa tagctgggatgtgtttggcaattggtttgaccttgcttcttggataaagtatatacaatatggaatttatgtagttgtaggagtaatactgttaagaat agtgatctatatagtacaaatgctagctaagttaaggcaggggtataggccagtgttctcttccccaccctcttatttccagtagactcatacccaa caggacccggcactgccaaccagagaaggcaaagaaggagacggtggagaaggcggtggatcctattatcgatccattgcatacgttgtat ccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggt cattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgac gtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagt acatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgg gactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacgtcaatgggcgtggatagcggtttg actcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaac tccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatccagctggc taccggtatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagtt cagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgaaccaccggcaagctgcccgtgccctg gcccaccctcgtgaccaccttcggctacggcgtgcagtgcttcgcccgctaccccgaccacatgcgccagcacgacttcttcaagtccgcca tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgac accctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagc cacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcag ctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctg agcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtac aagtaaggatcttaagcatgctcgcgaagcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgc tccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctg tctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccac cacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggg gctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcg cgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctt cgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgtttcgcctcgcgactcgagcaaactcacactcaagtttatttg caaactcacactcaagtttatttgcaaactcacactcaagtcatgcatggtcgagttttataaaagaaaaggggggactggaagggatttattac agtgcaagaagacatagaatcttagacatgtacttagaaaaggaagaaggcatcataccagattggcaggattacacctcaggaccaggaat tagatacccaaagacatttggctggctatggaaattagtccctgtaaatgtatcagatgaggcacaggaggatgagaggcattatttaatgcag ccagctcaaacttccaagtgggatgacccttggggagaggttctagcgtggaagtttgatccaactctagcctacacttatgaggcatatgcta gatacccagaagagttggaagcaagtcaggcctgtcagaactgcatttcgctctgtattcagtcgctctgcggagaggctggcagattgagcc ctgggaggttctctccagcactagcaggtagagcctgggtgttccctgctagactctcaccagcacttggccagtgctgggcagagtggctcc acgcttgcttgcttaaagacctcttcaataaagctgccattttagaagtaagccagtgtgtgttcccatctctcctagtcgccgcctggtcaactcg gtactcggtaataagaagaccctggtctgttaggaccctttctgctttgagaaaccgaagcaggaaaatccctagca

[0591] SEQ ID NO: 20: optimized pegRNA gggcacgggcagcttgccgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcg gtccagttcatctgtactaccggcaagctgccc

[0592] SEQ ID NO: 21: YFPs-IRES-Puro sequence atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtg tccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgaaccaccggcaagctgcccgtgccctggcccac cctcgtgaccaccttcggctacggcgtgcagtgcttcgcccgctaccccgaccacatgcgccagcacgacttcttcaagtccgccatgcccg aaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaa cgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgc cgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa gcttggatcccgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttc caccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaag gaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagc ggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccac gttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgt atgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtg gttttcctttgaaaaacacgatgataagcttgccacaacccacaaggagacgaccttccatgaccgagtacaagcccacggtgcgcctcgcc acccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgacccggacc gccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggc gccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagc ggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcg gcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccg ccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggacc gcgcacctggtgcatgacccgcaagcccggtgcctag

[0593] SEQ ID NO: 22: DNA sequence of GAG-PE V2 atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtgggcggccgcta atacgactcactatagggagagccgccaccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcg acaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaat tcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggc caaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaac atcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacct gcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtg gacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatc ctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctg attgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacg acgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgct gagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacct gaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctac attgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaag ctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccat tctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgg gccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagc ctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcg agcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcg agtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggaca aggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaa cggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccg gaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatg cagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacatt gccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggca caagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacc tgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcct cagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccga agaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggc cgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca cagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctgg tgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgg gaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaa gagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacg gcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcgg aaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagagg aacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctgg tggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcg agaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgag ctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttc ctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaag caccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtacttt gacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacga gacacggatcgacctgtctcagctgggaggtgactctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtc agcaacaccagagagcagtggcggcagcagcggcggcagcagcaccctaaatatagaagatgagtatcggctacatgagacctcaaaag agccagatgtttctctagggtccacatggctgtctgattttcctcaggcctgggcggaaaccgggggcatgggactggcagttcgccaagctc ctctgatcatacctctgaaagcaacctctacccccgtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagccccaca tacagagactgttggaccagggaatactggtaccctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgat tataggcctgtccaggatctgagagaagtcaacaagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggc tcccaccgtcccaccagtggtacactgtgcttgatttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgcctttga gtggagagatccagagatgggaatctcaggacaattgacctggaccagactcccacagggtttcaaaaacagtcccaccctgtttaatgagg cactgcacagagacctagcagacttccggatccagcacccagacttgatcctgctacagtacgtggatgacttactgctggccgccacttctg agctagactgccaacaaggtactcgggccctgttacaaaccctagggaacctcgggtatcgggcctcggccaagaaagcccaaatttgcca gaaacaggtcaagtatctggggtatcttctaaaagagggtcagagatggctgactgaggccagaaaagagactgtgatggggcagcctact ccgaagacccctcgacaactaagggagttcctagggaaggcaggcttctgtcgcctcttcatccctgggtttgcagaaatggcagcccccct gtaccctctcaccaaaccggggactctgtttaattggggcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgcccc agccctggggttgccagatttgactaagccctttgaactctttgtcgacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactggg accttggcgtcggccggtggcctacctgtccaaaaagctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattg ccgtactgacaaaggatgcaggcaagctaaccatgggacagccactagtcattctggccccccatgcagtagaggcactagtcaaacaacc ccccgaccgctggctttccaacgcccggatgactcactatcaggccttgcttttggacacggaccgggtccagttcggaccggtggtagccct gaacccggctacgctgctcccactgcctgaggaagggctgcaacacaactgccttgatgaaaattcatcaccctctggcggctcaaaaagaa ccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaa SEQ ID NO: 23: Amino acid sequence of GAG-PE V2

[0594] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGGRPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINA SGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE

[0595] ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLDENSSPSGGSKRTADGSEFEPKKKRKV SEQ ID NO: 24: DNA sequence of GAG-PE V3 atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggttctagactgcctcc acttgaaagactgacactgggatcattacaattacctcctttagaacgattaacactcggttcactacagcttccgcctcttgagagattgacatta gcgcggccgcaaacgcgtagttccctgtatccagccctcacacctgcgcggccgctaatacgactcactatagggagagccgccaccatga aacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacatcggcac caactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcat caagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatac accagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaag agtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtacc ccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaa gttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaac cagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctgga aaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaag agcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcgg cgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcacc aaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctg cctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctaca agttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcgg accttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaa ggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctg gatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagc ggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctg accaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaag accaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaag atcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacatt ctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaa agtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtcc ggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaaga ggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagg gcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccaga gagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagcca gatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtg gaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaaggacgactccatcgacaacaa ggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcgg cagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggc cggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacga cgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagt gcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctgga aagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgcca agtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa acggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtga aaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggac tgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaaga aactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaaggg ctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctg ccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaaggg ctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctcc aagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgag aatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagc accaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgac tctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcg gcggcagcagcaccctaaatatagaagatgagtatcggctacatgagacctcaaaagagccagatgtttctctagggtccacatggctgtctg attttcctcaggcctgggcggaaaccgggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctctaccccc gtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggaccagggaatactggtac cctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctgagagaagtcaac aagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctcccaccgtcccaccagtggtacactgtgcttga tttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgcctttgagtggagagatccagagatgggaatctcaggac aattgacctggaccagactcccacagggtttcaaaaacagtcccaccctgtttaatgaggcactgcacagagacctagcagacttccggatcc agcacccagacttgatcctgctacagtacgtggatgacttactgctggccgccacttctgagctagactgccaacaaggtactcgggccctgtt acaaaccctagggaacctcgggtatcgggcctcggccaagaaagcccaaatttgccagaaacaggtcaagtatctggggtatcttctaaaag agggtcagagatggctgactgaggccagaaaagagactgtgatggggcagcctactccgaagacccctcgacaactaagggagttcctag ggaaggcaggcttctgtcgcctcttcatccctgggtttgcagaaatggcagcccccctgtaccctctcaccaaaccggggactctgtttaattg gggcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgccccagccctggggttgccagatttgactaagccctttga actctttgtcgacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactgggaccttggcgtcggccggtggcctacctgtccaaa aagctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattgccgtactgacaaaggatgcaggcaagctaacca tgggacagccactagtcattctggccccccatgcagtagaggcactagtcaaacaaccccccgaccgctggctttccaacgcccggatgact cactatcaggccttgcttttggacacggaccgggtccagttcggaccggtggtagccctgaacccggctacgctgctcccactgcctgagga agggctgcaacacaactgccttgatatcctggccgaagcccacggaacccgacccgacctaacggaccagccgctcccagacgccgacc acacctggtacacggatggaagcagtctcttacaagagggacagcgtaaggcgggagctgcggtgaccaccgagaccgaggtaatctgg gctaaagccctgccagccgggacatccgctcagcgggctgaactgatagcactcacccaggccctaaagatggcagaaggtaagaagcta aatgtttatactgatagccgttatgcttttgctactgcccatatccatggagaaatatacagaaggcgtgggtggctcacatcagaaggcaaaga gatcaaaaataaagacgagatcttggccctactaaaagccctctttctgcccaaaagacttagcataatccattgtccaggacatcaaaaggga cacagcgccgaggctagaggcaaccggatggctgaccaagcggcccgaaaggcagccatcacagagactccagacacctctaccctcct catagaaaattcatcaccctctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaa

[0596] SEQ ID NO: 25: Amino acid sequence of GAG-PE V3

[0597] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP

[0598] DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP

[0599] LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA

[0600] PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN

[0601] PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT

[0602] QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP

[0603] NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG

[0604] DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV

[0605] VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL

[0606] YPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTLARPQTRSSLYPALTPARP

[0607] LIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKK

[0608] FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMA

[0609] KVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

[0610] RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL

[0611] SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD

[0612] DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL

[0613] LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN

[0614] REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA

[0615] RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY

[0616] EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC

[0617] FDSVEISGVEDRFNASLGTYHDLLI<III<DI<DFLDNEENEDILEDIVLTLTLFEDREMIEERL

[0618] KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM

[0619] QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH

[0620] KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLY

[0621] YLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE

[0622] EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

[0623] QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA

[0624] VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

[0625] LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP

[0626] KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER

[0627] SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

[0628] VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA

[0629] YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGL

[0630] YETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKE

[0631] PDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP

[0632] HIQRLLDQGILVPCQSPWNTPLLPVI<I<PGTNDYRPVQDLREVNI<RVEDIHPTVPNPYNLL

[0633] SGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP

[0634] TLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASA

[0635] KKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIP

[0636] GFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQ GYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWA KALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEG KEIKNKDEILALLKALFLPKRLSnHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST LLIENSSPSGGSKRTADGSEFEPKKKRKV

[0637] SEQ ID NO: 26: DNA sequence of PE-GAG V4 atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggttctagactgcctcc acttgaaagactgacactgggatcattacaattacctcctttagaacgattaacactcggttcactacagcttccgcctcttgagagattgacatta gcgcggccgcaaacgcgtagttccctgtatccagccctcacacctgcgcggccgctaatacgactcactatagggagagccgccaccatga aacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacatcggcac caactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcat caagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatac accagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaag agtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtacc ccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaa gttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaac cagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctgga aaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaag agcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcgg cgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcacc aaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctg cctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctaca agttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcgg accttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaa ggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctg gatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagc ggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctg accaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaag accaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaag atcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacatt ctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaa agtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtcc ggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaaga ggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagg gcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccaga gagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagcca gatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtg gaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaaggacgactccatcgacaacaa ggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcgg cagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggc cggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacga cgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagt gcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctgga aagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgcca agtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa acggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtga aaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggac tgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaaga aactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaaggg ctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctg ccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaaggg ctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctcc aagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgag aatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagc accaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgac tctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcg gcggcagcagcaccctaaatatagaagatgagtatcggctacatgagacctcaaaagagccagatgtttctctagggtccacatggctgtctg attttcctcaggcctgggcggaaaccgggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctctaccccc gtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggaccagggaatactggtac cctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctgagagaagtcaac aagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctcccaccgtcccaccagtggtacactgtgcttga tttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgcctttgagtggagagatccagagatgggaatctcaggac aattgacctggaccagactcccacagggtttcaaaaacagtcccaccctgtttaatgaggcactgcacagagacctagcagacttccggatcc agcacccagacttgatcctgctacagtacgtggatgacttactgctggccgccacttctgagctagactgccaacaaggtactcgggccctgtt acaaaccctagggaacctcgggtatcgggcctcggccaagaaagcccaaatttgccagaaacaggtcaagtatctggggtatcttctaaaag agggtcagagatggctgactgaggccagaaaagagactgtgatggggcagcctactccgaagacccctcgacaactaagggagttcctag ggaaggcaggcttctgtcgcctcttcatccctgggtttgcagaaatggcagcccccctgtaccctctcaccaaaccggggactctgtttaattg gggcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgccccagccctggggttgccagatttgactaagccctttga actctttgtcgacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactgggaccttggcgtcggccggtggcctacctgtccaaa aagctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattgccgtactgacaaaggatgcaggcaagctaacca tgggacagccactagtcattctggccccccatgcagtagaggcactagtcaaacaaccccccgaccgctggctttccaacgcccggatgact cactatcaggccttgcttttggacacggaccgggtccagttcggaccggtggtagccctgaacccggctacgctgctcccactgcctgagga agggctgcaacacaactgccttgatgaaaattcatcaccctctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaa gaagaggaaagtctaa

[0638] SEQ ID NO: 27: Amino acid sequence of PE-GAG V4

[0639] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN

[0640] PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV

[0641] VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTLARPQTRSSLYPALTPARP LIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKK

[0642] FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMA KVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

[0643] RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN

[0644] REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC FDSVEISGVEDRFNASLGTYHDLL1<III<DI<DFLDNEENEDILEDIVLTLTLFEDREMIEERL

[0645] KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLY YLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE

[0646] EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP

[0647] KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGL

[0648] YETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKE PDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP HIQRLLDQGILVPCQSPWNTPLLPVI<I<PGTNDYRPVQDLREVNI<RVEDIHPTVPNPYNLL SGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP

[0649] TLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASA KKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIP GFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQ GYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDENS SPSGGSKRTADGSEFEPKKKRKV

[0650] SEQ ID NO: 28: epegRNA yfps gggcacgggcagcttgccgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcg gtccagttcatctgtactaccggcaagctgcccatttaatccgcggttctatctagttacgcgttaaaccaactagaa

[0651] SEQ ID NO: 29: PegRNA YFPs TAR-trimmedEvoPreQi gggcacgggcagcttgccgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcg gtccagttcatctgtactaccggcaagctgcccactgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatctagt tacgcgttaaaccaactagaa

[0652] SEQ ID NO: 30: PegRNAYFPs MS2-trimmedEvoPreQi gggcacgggcagcttgccgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcg gtccagttcatctgtactaccggcaagctgcccactgttcaacatgaggatcacccatgtgaattcatttaatccgcggttctatctagttacgcgt taaaccaactagaa

[0653] SEQ ID NO: 31: PegRNA YFPs PP7-trimmedEvoPreQi gggcacgggcagcttgccgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcg gtccagttcatctgtactaccggcaagctgcccactgttcaactaaggagtttatatggaaacccttatttaatccgcggttctatctagttacgcgt taaaccaactagaa

[0654] SEQ ID NO: 32: DNA sequence of GAG-MCP atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtggaggtgcttcta actttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaacttcgctaacgggatcgctgaatggatca gctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgc ctaaaggcgcctggcgttcgtacttaaatatggaactaaccattccaattttcgccacgaattccgactgcgagcttattgttaaggcaatgcaag gtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctacgcggattcttcaactttcttgtacaaagtgggcgg aggttaa

[0655] SEQ ID NO: 33: Amino acid sequence of GAG-MCP

[0656] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGGGASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVR QSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIYADSSTFLYKVGGG

[0657] SEQ ID NO: 34: DNA sequence of GAG-PP7CP atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtatgctagcctcca aaaccatcgttctttcggtcggcgaggctactcgcactctgactgagatccagtccaccgcagaccgtcagatcttcgaagagaaggtcggg cctctggtgggtcggctgcgcctcacggcttcgctccgtcaaaacggagccaagaccgcgtatcgagtcaacctaaaactggatcaggcgg acgtcgttgattccggacttccgaaagtgcgctacactcaggtatggtcgcacgacgtgacaatcgttgcgaatagcaccgaggcctcgcgc aaatcgttgtacgatttgaccaagtccctcgtcgcgacctcgcaggtcgaagatcttgtcgtcaaccttgtgccgctgggccgttga SEQ ID NO: 35: Amino acid sequence of GAG-PP7CP

[0658] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGMLASKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAK TAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQ VEDLWNLVPLGR

[0659] SEQ ID NO: 36: DNA sequence of GAG-TAT1 atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtatggagccagtag atcctagactagagccctggaagcatccaggaagtcagcctaaaactgcttgtaccaattgctattgtaaaaagtgttgctttcattgccaagttt gtttcataacaaaagccttaggcatctcctatggcaggaagaagcggagacagcgacgaagacctcctcaaggcagtcagactcatcaagtt tctctatcaaagcaacccacctcccaatcccgaggggacccgacaggcccgaaggaatcgaagaagaaggtggagagagagacagaga cagatccattcgatggaggctga

[0660] SEQ ID NO: 37: Amino acid sequence of GAG-TAT1

[0661] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGMEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKK RRQRRRPPQGSQTHQVSLSKQPTSQSRGDPTGPKESKKKVERETETDPFDGG

[0662] SEQ ID NO: 38: DNA sequence of GAG-TATldelta2-26 atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtatgtgtaaaaagtg ttgctttcattgccaagtttgtttcataacaaaagccttaggcatctcctatggcaggaagaagcggagacagcgacgaagacctcctcaaggc agtcagactcatcaagtttctctatcaaagcaacccacctcccaatcccgaggggacccgacaggcccgaaggaatcgaagaagaaggtg gagagagagacagagacagatccattcgatggaggctga

[0663] SEQ ID NO: 39: Amino acid sequence of GAG-TATldelta2-26

[0664] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGMCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQPTSQS RGDPTGPKESKKKVERETETDPFDGG SEQ ID NO: 40: Peg RNA TAR-trimmedEvoPreQi HEK CTT insertion ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcgg tcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatctagttacgcgtt aaaccaactagaa

[0665] SEQ ID NO: 41: PegRNA TAR-trimmedEvoPreQi HEK3 l-5deletion ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcgg tcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatctagttacgcgtt aaaccaactagaa

[0666] SEQ ID NO: 42: PegRNA TAR-trimmedEvoPreQi RNF2 GTA insertion gtcatcttagtcattacctggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccgagtcggtcc aacgaacacctcagtacgtaatgactaagatgttcaacgccagatctgagcctgggagctctctggccctcgaggcgcggttctatctagttac gcgtt aaaccaactagaa

[0667] SEQ ID NO: 43: SWYM1MM atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtg tccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgaaccaccgggaagctgcccgtgccctggcccac cctcgtgaccaccttcggctacggcctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccg aaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaa cgtctatatcatggccgacaagcagaggaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgc cgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag

[0668] SEQ ID NO: 44: SWYM2MM atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtg tccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgaaccaccggcaagctcccagtgccctggcccacc ctcgtgaccaccttcggctacggcctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccga aggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggt gaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacg tctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccg accactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaag accccaacgagaagcgagatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag

[0669] SEQ ID NO: 45: ePegRNA sequence for CTT insertion (TAR-trimmed EvoPreQl)

[0670] Ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggacc gagtcggtcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatcta gttacgcgttaaaccaactagaatttttt SEQ ID NO: 46: t!7 mutant ggcccagactgagcacttgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccg agtcggtcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatctag ttacgcgttaaaccaactagaatttttt

[0671] SEQ ID NO: 47: tl4 mutant ggcccagactgagtacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccg agtcggtcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatctag ttacgcgttaaaccaactagaatttttt

[0672] SEQ ID NO: 48: tlO mutant ggcccagacggagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggacc gagtcggtcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatcta gttacgcgttaaaccaactagaatttttt

[0673] SEQ ID NO: 49: g6 mutant ggcccggactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggacc gagtcggtcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatcta gttacgcgttaaaccaactagaatttttt

[0674] SEQ ID NO: 50: t3 mutant ggtccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggaccg agtcggtcctctgccatcaaagcgtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatctag ttacgcgttaaaccaactagaatttttt

[0675] SEQ ID NO: 51: pAl mutant ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggacc gagtcggtcctctgccatcaaagagtgctcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatcta gttacgcgttaaaccaactagaatttttt

[0676] SEQ ID NO: 52: pA5 mutant ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggacc gagtcggtcctctgccatcaaagcgtgatcagtctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatcta gttacgcgttaaaccaactagaatttttt

[0677] SEQ ID NO: 53: pG9 mutant

[0678] Ggcccagactgagcacgtgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgggacc gagtcggtcctctgccatcaaagcgtgctcaggctgttcaacgccagatctgagcctgggagctctctggcatttaatccgcggttctatcta gttacgcgttaaaccaactagaatttttt SEQ ID NO: 54: DNAsequence of GAG-PEmax (vim) atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtgggcggccgcta atacgactcactatagggagagccgccaccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcg acaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaat tcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggc caaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaac atcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacct gcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtg gacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatc ctgtctgccagactgagcaagagcagaaagctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctg attgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacg acgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgct gagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacct gaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctac attgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaag ctgaagagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccat tctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgg gccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagc ctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcg agcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcg agtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggaca aggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaa cggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccg gaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatg cagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacatt gccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggca caagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacc tgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcct cagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccga agaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggc cgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca cagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctgg tgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgg gaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaa gagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacg gcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcgg aaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagagg aacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctgg tggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcg agaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgag ctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttc ctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaag caccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtacttt gacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacga gacacggatcgacctgtctcagctgggaggtgactccggcggaagctctggtggcagcaagcggaccgccgacggctctgaattcgaga gccctaagaagaaaagaaaggtgagcggaggctctagcggcggaagcaccctgaacattgaagacgagtatagactgcatgaaacaagc aaggaacccgacgtgtccctgggctccacctggctgtccgactttccccaggcctgggccgagacaggaggaatgggcctggccgtgcgg caggcacccctgatcatccctctgaaggccacctctacacccgtgagcatcaagcagtaccctatgtctcaggaggccagactgggcatcaa gcctcacatccagaggctgctggaccagggcatcctggtgccatgccagagcccctggaacacaccactgctgcccgtgaagaagccagg caccaatgactatagacccgtgcaggatctgagagaggtgaacaagagggtggaggatatccaccccaccgtgcccaacccttacaatctg ctgtccggcctgcccccttctcaccagtggtatacagtgctggacctgaaggatgccttcttttgtctgagactgcaccctaccagccagccact gttcgcctttgagtggagggaccctgagatgggcatctctggccagctgacctggacacgcctgcctcagggcttcaagaatagcccaacac tgtttaacgaggccctgcaccgcgacctggcagatttccggatccagcacccagatctgatcctgctgcagtacgtggacgatctgctgctgg ccgccaccagcgagctggattgccagcagggaacacgcgccctgctgcagaccctgggaaacctgggatatagggcatccgccaagaag gcccagatctgtcagaagcaggtgaagtacctgggctatctgctgaaggagggccagagatggctgacagaggccaggaaggagacagt gatgggccagccaacacccaagaccccaagacagctgagggagttcctgggcaaagcaggattttgcaggctgttcatcccaggattcgca gagatggcagcacctctgtacccactgaccaagccgggcaccctgtttaattggggccctgaccagcagaaggcctatcaggagatcaagc aggccctgctgacagcaccagccctgggcctgccagacctgaccaagcctttcgagctgtttgtggatgagaagcagggctacgccaagg gcgtgctgacccagaagctgggaccatggagacggcccgtggcctatctgtccaagaagctggacccagtggcagcaggatggccacca tgcctgaggatggtggcagcaatcgccgtgctgacaaaggatgccggcaagctgaccatgggacagccactggtcatcctggcaccacac gcagtggaggccctggtgaagcagcctccagatcgctggctgtctaacgcccggatgacacactaccaggccctgctgctggacaccgatc gcgtgcagtttggccctgtggtggccctgaatccagccaccctgctgcctctgccagaggagggcctgcagcacaactgtctggacatcctg gcagaggcacacggaacaaggccagacctgaccgatcagcccctgcctgacgccgatcacacatggtataccgatggaagctccctgctg caggagggccagaggaaggcaggagcagcagtgaccacagagacagaagtgatctgggccaaggccctgccagcaggcacatccgcc cagcgggccgagctgatcgccctgacccaggccctgaagatggccgagggcaagaagctgaacgtgtacacagactccagatatgccttc gccaccgcacacatccacggagagatctacaggcgccggggctggctgacctctgagggcaaggagatcaagaacaaggatgagatcct ggccctgctgaaggccctgtttctgcccaagcggctgagcatcatccactgtcctggacaccagaagggacactccgccgaggcaagggg caatcggatggccgaccaggccgccagaaaggctgctattactgaaactcccgacacttccactctgctgattgaaaactcctccccttctgg cggctcaaaaagaaccgccgacggcagcgaattcgagtctcccaagaagaagaggaaagtcggctctggccctgccgctaagagagtga agctggactaa

[0679] SEQ ID NO: 55: Amino acid sequence of GAG-PEmax (Vim)

[0680] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGGRPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINA SGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR

[0681] QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDE YRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHP TVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTR LPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLG KAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKP FELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDA GKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPA TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD

[0682] SEQ ID NO: 56: DNA sequence of GAG-PEmaxARNAseH (V2m) atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtgggcggccgcta atacgactcactatagggagagccgccaccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcg acaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaat tcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggc caaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaac atcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacct gcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtg gacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatc ctgtctgccagactgagcaagagcagaaagctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctg attgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacg acgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgct gagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacct gaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctac attgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaag ctgaagagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccat tctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgg gccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagc ctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcg agcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcg agtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggaca aggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaa cggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccg gaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatg cagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacatt gccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggca caagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacc tgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcct cagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccga agaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggc cgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca cagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctgg tgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgg gaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaa gagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacg gcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcgg aaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagagg aacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctgg tggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcg agaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgag ctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttc ctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaag caccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtacttt gacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacga gacacggatcgacctgtctcagctgggaggtgactccggcggaagctctggtggcagcaagcggaccgccgacggctctgaattcgaga gccctaagaagaaaagaaaggtgagcggaggctctagcggcggaagcaccctgaacattgaagacgagtatagactgcatgaaacaagc aaggaacccgacgtgtccctgggctccacctggctgtccgactttccccaggcctgggccgagacaggaggaatgggcctggccgtgcgg caggcacccctgatcatccctctgaaggccacctctacacccgtgagcatcaagcagtaccctatgtctcaggaggccagactgggcatcaa gcctcacatccagaggctgctggaccagggcatcctggtgccatgccagagcccctggaacacaccactgctgcccgtgaagaagccagg caccaatgactatagacccgtgcaggatctgagagaggtgaacaagagggtggaggatatccaccccaccgtgcccaacccttacaatctg ctgtccggcctgcccccttctcaccagtggtatacagtgctggacctgaaggatgccttcttttgtctgagactgcaccctaccagccagccact gttcgcctttgagtggagggaccctgagatgggcatctctggccagctgacctggacacgcctgcctcagggcttcaagaatagcccaacac tgtttaacgaggccctgcaccgcgacctggcagatttccggatccagcacccagatctgatcctgctgcagtacgtggacgatctgctgctgg ccgccaccagcgagctggattgccagcagggaacacgcgccctgctgcagaccctgggaaacctgggatatagggcatccgccaagaag gcccagatctgtcagaagcaggtgaagtacctgggctatctgctgaaggagggccagagatggctgacagaggccaggaaggagacagt gatgggccagccaacacccaagaccccaagacagctgagggagttcctgggcaaagcaggattttgcaggctgttcatcccaggattcgca gagatggcagcacctctgtacccactgaccaagccgggcaccctgtttaattggggccctgaccagcagaaggcctatcaggagatcaagc aggccctgctgacagcaccagccctgggcctgccagacctgaccaagcctttcgagctgtttgtggatgagaagcagggctacgccaagg gcgtgctgacccagaagctgggaccatggagacggcccgtggcctatctgtccaagaagctggacccagtggcagcaggatggccacca tgcctgaggatggtggcagcaatcgccgtgctgacaaaggatgccggcaagctgaccatgggacagccactggtcatcctggcaccacac gcagtggaggccctggtgaagcagcctccagatcgctggctgtctaacgcccggatgacacactaccaggccctgctgctggacaccgatc gcgtgcagtttggccctgtggtggccctgaatccagccaccctgctgcctctgccagaggagggcctgcagcacaactgtctggaaaactcc tccccttctggcggctcaaaaagaaccgccgacggcagcgaattcgagtctcccaagaagaagaggaaagtcggctctggccctgccgct aagagagtgaagctggactaa

[0683] SEQ ID NO: 57: Amino acid sequence of GAG-PEmaxARNAseH (V2m)

[0684] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGGRPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINA SGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR

[0685] QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDE YRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHP TVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTR LPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLG KAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKP FELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDA GKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPA TLLPLPEEGLQHNCLENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD

[0686] SEQ ID NO: 58: DNA sequence of GAG-3NES-Pro-PEmax (V3m) atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggttctagactgcctcc acttgaaagactgacactgggatcattacaattacctcctttagaacgattaacactcggttcactacagcttccgcctcttgagagattgacatta gcgcggccgcaaacgcgtagttccctgtatccagccctcacacctgcgctaccgcggccgctaatacgactcactatagggagagccgcca ccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacat cggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggc acagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaag aagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacaga ctggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgag aagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccaca tgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagac ctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagaaa gctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaac ttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccag atcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgaga tcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagc agctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagtt ctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaagagagaggacctgctgcggaagca gcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattc ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattc gcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatc gagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacg agctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgt tcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgt ggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgag gacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgac gacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagc agtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgaccttta aagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaag aagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggc cagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggca gccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgta cgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaaggacgactccatcgaca acaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactg gcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggata aggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagt acgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttaca aagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagc tggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctacc gccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgag acaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatc gtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaa ggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtc caagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagcc aagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctgg cctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctg aagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgag ttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcagg ccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtaca ccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggag gtgactccggcggaagctctggtggcagcaagcggaccgccgacggctctgaattcgagagccctaagaagaaaagaaaggtgagcgg aggctctagcggcggaagcaccctgaacattgaagacgagtatagactgcatgaaacaagcaaggaacccgacgtgtccctgggctccac ctggctgtccgactttccccaggcctgggccgagacaggaggaatgggcctggccgtgcggcaggcacccctgatcatccctctgaaggc cacctctacacccgtgagcatcaagcagtaccctatgtctcaggaggccagactgggcatcaagcctcacatccagaggctgctggaccag ggcatcctggtgccatgccagagcccctggaacacaccactgctgcccgtgaagaagccaggcaccaatgactatagacccgtgcaggat ctgagagaggtgaacaagagggtggaggatatccaccccaccgtgcccaacccttacaatctgctgtccggcctgcccccttctcaccagtg gtatacagtgctggacctgaaggatgccttcttttgtctgagactgcaccctaccagccagccactgttcgcctttgagtggagggaccctgag atgggcatctctggccagctgacctggacacgcctgcctcagggcttcaagaatagcccaacactgtttaacgaggccctgcaccgcgacct ggcagatttccggatccagcacccagatctgatcctgctgcagtacgtggacgatctgctgctggccgccaccagcgagctggattgccagc agggaacacgcgccctgctgcagaccctgggaaacctgggatatagggcatccgccaagaaggcccagatctgtcagaagcaggtgaag tacctgggctatctgctgaaggagggccagagatggctgacagaggccaggaaggagacagtgatgggccagccaacacccaagaccc caagacagctgagggagttcctgggcaaagcaggattttgcaggctgttcatcccaggattcgcagagatggcagcacctctgtacccactg accaagccgggcaccctgtttaattggggccctgaccagcagaaggcctatcaggagatcaagcaggccctgctgacagcaccagccctg ggcctgccagacctgaccaagcctttcgagctgtttgtggatgagaagcagggctacgccaagggcgtgctgacccagaagctgggaccat ggagacggcccgtggcctatctgtccaagaagctggacccagtggcagcaggatggccaccatgcctgaggatggtggcagcaatcgcc gtgctgacaaaggatgccggcaagctgaccatgggacagccactggtcatcctggcaccacacgcagtggaggccctggtgaagcagcct ccagatcgctggctgtctaacgcccggatgacacactaccaggccctgctgctggacaccgatcgcgtgcagtttggccctgtggtggccct gaatccagccaccctgctgcctctgccagaggagggcctgcagcacaactgtctggacatcctggcagaggcacacggaacaaggccag acctgaccgatcagcccctgcctgacgccgatcacacatggtataccgatggaagctccctgctgcaggagggccagaggaaggcagga gcagcagtgaccacagagacagaagtgatctgggccaaggccctgccagcaggcacatccgcccagcgggccgagctgatcgccctga cccaggccctgaagatggccgagggcaagaagctgaacgtgtacacagactccagatatgccttcgccaccgcacacatccacggagag atctacaggcgccggggctggctgacctctgagggcaaggagatcaagaacaaggatgagatcctggccctgctgaaggccctgtttctgc ccaagcggctgagcatcatccactgtcctggacaccagaagggacactccgccgaggcaaggggcaatcggatggccgaccaggccgc cagaaaggctgctattactgaaactcccgacacttccactctgctgattgaaaactcctccccttctggcggctcaaaaagaaccgccgacgg cagcgaattcgagtctcccaagaagaagaggaaagtcggctctggccctgccgctaagagagtgaagctggactaa

[0687] SEQ ID NO: 59: Amino acid sequence of GAG-3NES-Pro-PEmax (V3m)

[0688] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP

[0689] NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL

[0690] YPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTLARPQTRSSLYPALTPALP RPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA ILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY

[0691] DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK LKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE

[0692] RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY

[0693] LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI

[0694] MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG WLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAIT ETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD

[0695] SEQ ID NO: 60: DNA sequence of GAG-3NES-Pro-PEmaxARNAseH (V4m) atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggttctagactgcctcc acttgaaagactgacactgggatcattacaattacctcctttagaacgattaacactcggttcactacagcttccgcctcttgagagattgacatta gcgcggccgcaaacgcgtagttccctgtatccagccctcacacctgcgctaccgcggccgctaatacgactcactatagggagagccgcca ccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacat cggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggc acagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaag aagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacaga ctggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgag aagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccaca tgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagac ctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagaaa gctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaac ttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccag atcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgaga tcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagc agctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagtt ctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaagagagaggacctgctgcggaagca gcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattc ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattc gcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatc gagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacg agctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgt tcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgt ggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgag gacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgac gacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagc agtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgaccttta aagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaag aagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggc cagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggca gccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgta cgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaaggacgactccatcgaca acaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactg gcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggata aggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagt acgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttaca aagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagc tggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctacc gccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgag acaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatc gtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaa ggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtc caagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagcc aagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctgg cctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctg aagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgag ttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcagg ccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtaca ccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggag gtgactccggcggaagctctggtggcagcaagcggaccgccgacggctctgaattcgagagccctaagaagaaaagaaaggtgagcgg aggctctagcggcggaagcaccctgaacattgaagacgagtatagactgcatgaaacaagcaaggaacccgacgtgtccctgggctccac ctggctgtccgactttccccaggcctgggccgagacaggaggaatgggcctggccgtgcggcaggcacccctgatcatccctctgaaggc cacctctacacccgtgagcatcaagcagtaccctatgtctcaggaggccagactgggcatcaagcctcacatccagaggctgctggaccag ggcatcctggtgccatgccagagcccctggaacacaccactgctgcccgtgaagaagccaggcaccaatgactatagacccgtgcaggat ctgagagaggtgaacaagagggtggaggatatccaccccaccgtgcccaacccttacaatctgctgtccggcctgcccccttctcaccagtg gtatacagtgctggacctgaaggatgccttcttttgtctgagactgcaccctaccagccagccactgttcgcctttgagtggagggaccctgag atgggcatctctggccagctgacctggacacgcctgcctcagggcttcaagaatagcccaacactgtttaacgaggccctgcaccgcgacct ggcagatttccggatccagcacccagatctgatcctgctgcagtacgtggacgatctgctgctggccgccaccagcgagctggattgccagc agggaacacgcgccctgctgcagaccctgggaaacctgggatatagggcatccgccaagaaggcccagatctgtcagaagcaggtgaag tacctgggctatctgctgaaggagggccagagatggctgacagaggccaggaaggagacagtgatgggccagccaacacccaagaccc caagacagctgagggagttcctgggcaaagcaggattttgcaggctgttcatcccaggattcgcagagatggcagcacctctgtacccactg accaagccgggcaccctgtttaattggggccctgaccagcagaaggcctatcaggagatcaagcaggccctgctgacagcaccagccctg ggcctgccagacctgaccaagcctttcgagctgtttgtggatgagaagcagggctacgccaagggcgtgctgacccagaagctgggaccat ggagacggcccgtggcctatctgtccaagaagctggacccagtggcagcaggatggccaccatgcctgaggatggtggcagcaatcgcc gtgctgacaaaggatgccggcaagctgaccatgggacagccactggtcatcctggcaccacacgcagtggaggccctggtgaagcagcct ccagatcgctggctgtctaacgcccggatgacacactaccaggccctgctgctggacaccgatcgcgtgcagtttggccctgtggtggccct gaatccagccaccctgctgcctctgccagaggagggcctgcagcacaactgtctggaaaactcctccccttctggcggctcaaaaagaacc gccgacggcagcgaattcgagtctcccaagaagaagaggaaagtcggctctggccctgccgctaagagagtgaagctggactaa

[0696] SEQ ID NO: 61: Amino acid sequence of GAG-3NES-Pro-PEmaxARNAseH (V4m)

[0697] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV

[0698] VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTLARPQTRSSLYPALTPALP RPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA ILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD

[0699] LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK LKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP LARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY

[0700] LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK

[0701] VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQ GFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD

[0702] SEQ ID NO: 62: DNA sequence of GAG-PEmax-Pro-3NES (Vlmp) atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctgggaagctatagcagtagaccccc ctccctgggtcagacccttcgtgcaccctaaacctcccctctctcttcccccttcagccccctctctcccacctgaacccccactctcgaccccg ccccagtcctccctctatccggctctcacttctcctttaaacaccaaacctaggcctcaagtccttcctgatagcggaggaccactcattgatcta ctcacggaggaccctccgccttaccgggacccagggccaccctctcctgacgggaacggcgatagcggagaagtggcccctacagaagg agcccctgacccttccccaatggtatcccgcctgcggggaagaaaagaaccccccgtggcggattctactacctctcaggcgttcccccttc gcctgggagggaatggacagtatcaatactggccattttcctcctctgacctctataactggaaaaataacaacccctctttctccgaggaccca gctaaattgacagctttgatcgagtccgttctccttactcatcagcccacttgggatgactgccaacagctattagggaccctgctgacgggag aagaaaaacagcgagtgctcctagaggcccgaaaggcggttcgaggggaggacggacgcccaactcagctgcccaatgacattaatgat gcttttcccttggaacgtcccgactgggactacaacacccaacgaggtaggaaccacctagtccactatcgccagttgctcctagcgggtctc caaaacgcgggcagaagccccaccaatttggccaaggtaaaagggataacccagggacctaatgagtctccctcagcctttttagagagact caaggaggcctatcgcagatacactccttatgaccctgaggacccagggcaagaaaccaatgtggccatgtcattcatctggcagtccgccc cggatatcgggcgaaagttagagcggttagaagatttgaagagtaagaccttaggagacttagtgagggaagctgaaaagatctttaataaac gagaaaccccggaagaaagagaggaacgtattaggagagaaacagaggaaaaggaagaacgccgtagggcagaggatgtgcagaga gagaaggagagggaccgcagaagacatagagaaatgagtaagttgctggctactgtcgttagcgggcagagacaggatagacagggag gagagcgaaggaggccccaactcgaccacgaccagtgtgcctactgcaaagaaaagggacattgggctagagattgccccaagaagcca agaggaccccggggaccacgaccccaggcctccctcctgacgcgtagttccctgtatccagccctcacacctaccggtgggcggccgcta atacgactcactatagggagagccgccaccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcg acaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaat tcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggc caaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaac atcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacct gcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtg gacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatc ctgtctgccagactgagcaagagcagaaagctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctg attgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacg acgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgct gagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacct gaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctac attgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaag ctgaagagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccat I l l tctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgg gccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagc ctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcg agcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcg agtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggaca aggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaa cggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccg gaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatg cagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacatt gccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggca caagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacc tgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcct cagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccga agaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggc cgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca cagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctgg tgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgg gaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaa gagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacg gcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcgg aaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagagg aacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctgg tggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcg agaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgag ctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttc ctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaag caccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtacttt gacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacga gacacggatcgacctgtctcagctgggaggtgactccggcggaagctctggtggcagcaagcggaccgccgacggctctgaattcgaga gccctaagaagaaaagaaaggtgagcggaggctctagcggcggaagcaccctgaacattgaagacgagtatagactgcatgaaacaagc aaggaacccgacgtgtccctgggctccacctggctgtccgactttccccaggcctgggccgagacaggaggaatgggcctggccgtgcgg caggcacccctgatcatccctctgaaggccacctctacacccgtgagcatcaagcagtaccctatgtctcaggaggccagactgggcatcaa gcctcacatccagaggctgctggaccagggcatcctggtgccatgccagagcccctggaacacaccactgctgcccgtgaagaagccagg caccaatgactatagacccgtgcaggatctgagagaggtgaacaagagggtggaggatatccaccccaccgtgcccaacccttacaatctg ctgtccggcctgcccccttctcaccagtggtatacagtgctggacctgaaggatgccttcttttgtctgagactgcaccctaccagccagccact gttcgcctttgagtggagggaccctgagatgggcatctctggccagctgacctggacacgcctgcctcagggcttcaagaatagcccaacac tgtttaacgaggccctgcaccgcgacctggcagatttccggatccagcacccagatctgatcctgctgcagtacgtggacgatctgctgctgg ccgccaccagcgagctggattgccagcagggaacacgcgccctgctgcagaccctgggaaacctgggatatagggcatccgccaagaag gcccagatctgtcagaagcaggtgaagtacctgggctatctgctgaaggagggccagagatggctgacagaggccaggaaggagacagt gatgggccagccaacacccaagaccccaagacagctgagggagttcctgggcaaagcaggattttgcaggctgttcatcccaggattcgca gagatggcagcacctctgtacccactgaccaagccgggcaccctgtttaattggggccctgaccagcagaaggcctatcaggagatcaagc aggccctgctgacagcaccagccctgggcctgccagacctgaccaagcctttcgagctgtttgtggatgagaagcagggctacgccaagg gcgtgctgacccagaagctgggaccatggagacggcccgtggcctatctgtccaagaagctggacccagtggcagcaggatggccacca tgcctgaggatggtggcagcaatcgccgtgctgacaaaggatgccggcaagctgaccatgggacagccactggtcatcctggcaccacac gcagtggaggccctggtgaagcagcctccagatcgctggctgtctaacgcccggatgacacactaccaggccctgctgctggacaccgatc gcgtgcagtttggccctgtggtggccctgaatccagccaccctgctgcctctgccagaggagggcctgcagcacaactgtctggacatcctg gcagaggcacacggaacaaggccagacctgaccgatcagcccctgcctgacgccgatcacacatggtataccgatggaagctccctgctg caggagggccagaggaaggcaggagcagcagtgaccacagagacagaagtgatctgggccaaggccctgccagcaggcacatccgcc cagcgggccgagctgatcgccctgacccaggccctgaagatggccgagggcaagaagctgaacgtgtacacagactccagatatgccttc gccaccgcacacatccacggagagatctacaggcgccggggctggctgacctctgagggcaaggagatcaagaacaaggatgagatcct ggccctgctgaaggccctgtttctgcccaagcggctgagcatcatccactgtcctggacaccagaagggacactccgccgaggcaagggg caatcggatggccgaccaggccgccagaaaggctgctattactgaaactcccgacacttccactctgctgattgaaaactcctccccttctgg cggctcaaaaagaaccgccgacggcagcgaattcgagtctcccaagaagaagaggaaagtcggctctggccctgccgctaagagagtga agctggacaagcttacgcgtagttccctgtatccagccctcacacctaccggttctagactgcctccacttgaaagactgacactgggatcatta caattacctcctttagaacgattaacactcggttcactacagcttccgcctcttgagagattgacattataa

[0703] SEQ ID NO: 63: Amino acid sequence of GAG-PEmax-Pro-3NES (Vlmp)

[0704] MGQAVTTPLSLTLDHWKDVERTAHNLSVEVRKRRWVTFCSAEWPTFNVGWPRDGTFNP DIITQVKIKVFSPGPHGHPDQVPYIVTWEAIAVDPPPWVRPFVHPKPPLSLPPSAPSLPPEPP LSTPPQSSLYPALTSPLNTKPRPQVLPDSGGPLIDLLTEDPPPYRDPGPPSPDGNGDSGEVA PTEGAPDPSPMVSRLRGRKEPPVADSTTSQAFPLRLGGNGQYQYWPFSSSDLYNWKNNN PSFSEDPAKLTALIESVLLTHQPTWDDCQQLLGTLLTGEEKQRVLLEARKAVRGEDGRPT QLPNDINDAFPLERPDWDYNTQRGRNHLVHYRQLLLAGLQNAGRSPTNLAKVKGITQGP NESPSAFLERLKEAYRRYTPYDPEDPGQETNVAMSFIWQSAPDIGRKLERLEDLKSKTLG DLVREAEKIFNKRETPEEREERIRRETEEKEERRRAEDVQREKERDRRRHREMSKLLATV VSGQRQDRQGGERRRPQLDHDQCAYCKEKGHWARDCPKKPRGPRGPRPQASLLTRSSL YPALTPTGGRPLIRLTIGRAATMKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVI TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINA SGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL

[0705] QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF

[0706] SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDE YRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHP TVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTR LPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLG KAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKP FELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDA GKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPA TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLDKLTRSSLYP ALTPTGSRLPPLERLTLGSLQLPPLERLTLGSLQLPPLERLTL

[0707] SEQ ID NO: 64: GAG-PEmaxARNaseH-Pro-3NES (V2mp) atgggccaggctgttaccacccccttaagtttgactttagaccactggaaggatgtcgaacggacagcccacaacctgtcggtagaggttaga aaaaggcgctgggttacattctgctctgcagaatggccaaccttcaacgtcggatggccacgagacggcacttttaacccagacattattacac aggttaagatcaaggtcttctcacctggcccacatggacatccggatcaggtcccctacatcgtgacctg...

Claims

CLAIMS1. A virus-derived particle comprising a fusion protein comprising, a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and optionally a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity;2. The virus-derived particle according to claim 1, further comprising at least one prime editing guide RNA (pegRNA), in particular a pegRNA bound to the napDNAbp.

3. The virus-derived particle according to claim 1 or 2, wherein the virus-derived assembly protein is a virus-derived GAG protein.

4. The virus-derived particle according to any one of claims 1 to 3, wherein the cleavable linker contains three nuclear export signals.

5. The virus-derived particle according to any one of the preceding claims, wherein the fusion protein contains, from N-ter to C-ter, the virus-derived assembly protein, the prime editor and the cleavable linker.

6. The virus-derived particle according to any one of the preceding claims, wherein the protease cleavage sites are selected from Moloney murine leukemia virus (MMLV) protease cleavage sites such as MA / P12, pl2 / CA, CA / NC, NC / PR, RT / IN, or pl2E / p2E, and from Friend murine leukemia virus (FMLV) protease cleavage sites.

7. The virus-derived particle according to any one of the preceding claims, wherein the napDNAbp is a caspase, in particular is a Cas9, more particularly is a Cas9 nickase.

8. The virus-derived particle according to any one of the preceding claims, wherein the domain comprising a RNA-dependent DNA polymerase activity is a reverse transcriptase, inparticular is a MMLV reverse transcriptase or a Schizosaccharomyces pombe Tfl retrotransposon reverse transcriptase.

9. The virus-derived particle according to claim 8, wherein the reverse transcriptase does not contain a RNaseH domain.

10. The virus-derived particle according to any one of the preceding claims, wherein the prime editor is selected from PE2, PE3, PE4, PE5, PE6, NuPE, TwinPE and PEmax, in particular the prime editor is PE2; PEmax or PE6, such as PE6b, PE6c and PE6d, in particular PE6b.

11. The virus-derived particle according to any one of claims 2 to 10, wherein the pegRNA comprises two stem-loop structures in 3’, in particular two stem-loop structures selected from tEvoPreQi, CSY4, MS2, TAR, and PP7, Box C / D, Box B or the histone mRNA stem loop, more particularly selected from tEvPreQi, CSY4, MS2, TAR and PP7, more particularly selected from tEvQl, CSY4, TAR and PP7.

12. The virus-derived particle according to any one of claims 2 to 11, comprising:- a fusion protein containing, from N-ter to C-ter, the virus-derived assembly protein, the prime editor and the cleavable linker,- a prime editor that is PEmax or PE6, in particular PE6b, PE6c or PE6d, more particularly PE6b, and- a pegRNA comprising two stem-loop structures in 3’, in particular selected from tEvQl, CSY4, TAR and PP7.

13. The virus-derived particle according to any one of claims 2 to 12, comprising at least two pegRNAs.

14. The virus-derived particle according to any one of claims 2 to 13, further comprising at least one target molecule that binds to pegRNA, in particular that binds to one or more of the stem-loop structures of the pegRNA, in particular wherein the target molecule is selected from the group consisting of MCP, PP7CP, TAT, Csy4 (wild type or H29A mutant), L7Ae, LambdaN, SLBP, and Com, in particular from the group consisting of MCP, PP7CP, and TAT, more particularly the target molecule is a PP7CP or is a TAT.

15. The virus-derived particle according to any one of the preceding claims, further comprising one or more additional protein(s), in particular selected from reporter proteins, from helper proteins, and from DNA and / or RNA binding proteins, more particularly selected from the group consisting of fluorescent proteins such as mCherry, GFP, mKeita or luciferase; transcription factors; proteins limiting the expression of a surface cell marker in recipient cells; proteins boosting expression of a cell surface marker in recipient cells; proteins generating a selectable phenotype such as proteins inducing a resistance to a drug; recombinases such as CRE; integrases such as BxBl; internal proteins facilitating the incorporation of specific fusogens or viral envelopes; helper proteins including hMLHldn, P53dd, P65, Rad51, T5 exonuclease and FEN1 or binding proteins including MS2 coat protein, PP7 Coat protein, and TATI from HIV.

16. The virus-derived particle according to any one of the preceding claims, which is a retrovirus-derived particle, in particular is a gamma-retrovirus-derived particle.

17. The virus-derived particle according to any one of the preceding claims, further comprising one or more viral structural protein(s) or viral envelope protein(s), in particular comprising a VSV-G protein, a BAEV envelope or the R-less version of BAEV envelope (BRL), and human syncytin 1 (h-synl) in particular comprising a VSV-G protein, the R-less version of BAEV envelope (BRL) and h-synl.

18. A plurality of polynucleotides comprising:(i) one or more polynucleotide(s) encoding a fusion protein comprising: a virus-derived assembly protein, a cleavable linker containing, from N-ter to C-ter, a first protease cleavage site (PS), at least one nuclear export signal (NES) and a second protease cleavage site (PS), wherein the first and second protease cleavage sites may be different or identical, in particular are identical, and a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising a RNA-dependent DNA polymerase activity;(ii) a polynucleotide encoding a prime editing guide RNA (pegRNA), in particular a pegRNA bound to the napDNAbp, and(iii) optionally a polynucleotide encoding one or more pseudotyping viral envelope protein(s).

19. The plurality of polynucleotides of claim 18, wherein the pegRNA is contained in an intron under the control of a Pol II promoter, in particular of a Pol II CMV promoter.

20. The plurality of polynucleotides of claim 18 or 19, wherein the pegRNA is flanked on either side by a protease cleaving site, in particular by a csy4 protease cleaving site.

21. A method for altering a target nucleic acid comprising at least a target sequence in an eukaryotic cell, comprising the steps of: a) bringing into contact the said eukaryotic cell with a virus-derived particle according to any one of claims 1 to 17, or with a plurality of polynucleotides according to any one of claims 18 to 20, and b) collecting the said eukaryotic cell having an altered target nucleic acid.

Images