DNA-encoded fusion proteins for incorporation of prime editing protein / RNA complexes in virus-like particles

Abstract

The present invention relates to a virus-like particle (VLP) comprising a prime editing (PE) system, wherein the nuclease of the PE system is fused to a viral envelope protein, such as VSV-G; and / or a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N-terminal end of the Gag containing polypeptide. The invention furthermore relates to packaging systems, methods of producing such VLPs, fusion proteins, and uses thereof.

Description

[0001] DNA-ENCODED FUSION PROTEINS FOR INCORPORATION OF PRIME EDITING PROTEIN / RNA COMPLEXES IN VIRUS-LIKE PARTICLES

[0002] Technical field of the invention

[0003] The present invention relates to improved prime editing constructs for delivering via virus-like particles (VLPs). In particular, the present invention relates to prime editing constructs wherein the nuclease part of the construct is fused to part of the VLP. The invention also relates to packaging systems for producing such VLPs, methods of producing such VLPs, and uses thereof.

[0004] Background of the invention

[0005] CRISPR / Cas9-based genome editing has the potential to transform the lives of patients suffering from severe genetic diseases1. Editing of disease-causing genes using CRISPR / Cas9 is typically based on targeted DNA cleavage mediated by the Cas9 endonuclease directed by a single guide RIMA (sgRNA) to a predetermined locus. Repair of the DNA double-stranded break (DSB) by non-homologous end joining (NHEJ) leads to indel formation, whereas repair by homology-directed repair (HDR) supports precise editing2. However, this strategy suffers from low efficacy in primary cells as well as the need for co-administering a DNA repair template. Also, genotoxicity is a significant concern due to the formation of DSBs in both on- and off-target genomic loci.

[0006] Due to such challenges of conventional Cas9-based gene editing, alternative genome editing approaches involving neither formation of DSBs nor repair using a donor sequence have been developed. A novel technique called 'prime editing' facilitates genomic incorporation of the edit sequence copied from the prime editing guide RNA (pegRNA)3(Figure 1). Editing by prime editing is facilitated by the prime editor (PE), a fusion protein consisting of a Cas9 nickase fused to the reverse transcriptase derived from Moloney murine leukaemia virus (M-MLV)3. As prime editing relies on the administration of a larger fusion protein, therapeutic intervention is challenged by the restricted packaging capacity of conventional gene vehicles4. Such vehicles include lentiviral vectors and vectors based on adeno-associated virus (AAV)-derived vectors, both of which deliver the genes encoding PE and pegRNA, but delivery is in both cases challenged by the size of the gene cassettes, which are larger than the capacity of the vectors. Also, we have previously shown delivery of PE- and pegRNA-encoding gene cassettes in transfected DNA transposon vectors, which can be designed to accommodate the full-length gene cassettes for prime editing5. Effective genome editing therapies rely on delivery tools supporting short-lived activity of Cas9 / sgRNA complexes to restrict unintended modifications of the genome.

[0007] Contemporary virus-based delivery approaches, including the vector types mentioned above, facilitate delivery of Cas9 and sgRNA gene expression cassettes and potentially causes prolonged cellular exposure to active Cas9 / sgRNA complexes, which may lead to DNA cleavage in unwanted off-target genomic loci and genotoxicity. Genome editing can be achieved by DNA-free delivery of CRISPR / Cas9 ribonucleoprotein (RNP) complexes consisting of recombinant Cas9 protein complexed with synthetic sgRNAs6. Although RNPs are often used for effective ex vivo modification of cells by nucleofection, this method does not comply with in vivo use, and alternative methodologies are warranted for in vivo delivery of RNPs. Over the past few years, retro- and lentivirus-derived virus particles have been engineered to accommodate and transiently deliver foreign proteins. Our group has shown delivery of genome-modifying proteins, like DNA transposases7-9, zinc-finger nucleases (ZFN)10'11, TAL-effector nucleases10, and Cas912in lentivirus-derived nanoparticles (LVNPs). Also, more recently, we have demonstrated incorporation of Cas9 / sgRNA complexes in LVNPs, leading to potent targeted DNA cleavage in recipient cells.

[0008] WO2023 / 102538 Al discloses virus-like particles (VLPs) for delivering prime editors, and systems comprising such prime editor (PE) VLPs.

[0009] US 2023 / 0227793 Al discloses virus-like particles (eVLPs), comprising a membrane comprising a phospholipid bilayer with one or more virally-derived glycoproteins on the external side; and a cargo disposed in the core of the eVLP on the inside of the membrane, wherein the eVLP does not comprise an exogenous gag / pol protein. Hence, an improved method for prime editing genome-editing would be advantageous, and in particular more efficient and / or reliable particles for prime editing, would be advantageous.

[0010] Summary of the invention

[0011] Despite previous evidence for packaging of DNA-modifying enzymes in VLPs, the size of the PE-pegRNA complex is significantly larger than proteins and protein / RIMA complexes, which have previously been delivered in VLPs. Therefore, we developed new strategies for incorporating PE / pegRNA RNPs in VLPs.

[0012] We explored four different configurations for fusing PE (complexed during VLP production with pegRNA) to viral proteins. We refer to these configurations as GagPro-PEmax, PH-GagPro-PEmax, PH-Gag-PEmax, and VSV-G-PEmax, and demonstrate the capacity of such fusion strategies to support prime editing in cells treated with PE / pegRNA-loaded VLPs. The capacity of VLPs to accommodate and deliver PE / pegRNA is illustrated schematically in Figure 2.

[0013] Our findings establish VLP-directed prime editing and demonstrate the engineering of lentiviral particles to become 'true' genomic drugs that edit the genome without creation of DSBs or indels and without the need for supplying a repair template for HDR. These data mark the first examples of PE delivery by exploiting enveloped particles based on HIV-1 biology for RNP delivery in mammalian cells and expand the options for effective and safe delivery of CRISPR therapeutics.

[0014] In sum, the inventing team has identified improved methods of delivering prime editing (PE) systems / constructs to cells via optimized Lentivirus-Derived NanoParticles (LVNP) or VLPs, which provide several improvements compared to known methods. A prime editing system is schematically outlined in figure 1. In figure 2, a method of VLP generation in a producer cell and delivery to a target cell is outlined. The schematic representations in figures 1 and 2 shall be seen as specific embodiments of the invention and not a limitation to the overall concept of the present invention. In the example section the inventing team shows using different constructs and targets, that the invention is not limited to specific targets, but is to be considered a platform technology suitable to be adapted to a target desired by the end-user.

[0015] Example 1 provides an overview of the different prime editing constructs according to the invention and how they can be produced.

[0016] Example 2 demonstrates the capacity of VLPs to deliver larger genome editing toolkits including prime editing RNPs consisting of PEmax (with a size of 230 kDa) complexed with epegRNA. In example 2, the prime editing RNP is fused to the C- terminal of GagPro.

[0017] Example 3 demonstrates that addition of a Pleckstrin homology (PH) domain to the GagPro-PEmax configuration supports production of VLPs showing higher levels of prime editing in recipient cells. These findings suggest that the capacity of the PH domain to bind phosphatidylinositol in the plasma membrane supports better recruitment of the GagPro-PEmax polypeptide allowing potentially more PH- GagPro-PEmax polypeptides to be packaged in each VLP unit.

[0018] Example 4 shows effective prime editing in cells treated with VLPs packaged with PH-Gag-PEmax polypeptides. This demonstrates that protease derived from normal GagPol polypeptides, which are co-incorporated into the VLPs during production, can act on PH-Gag-PEmax and support release of PEmax / pegRNA RNPs during VLP maturation after budding. Thus, a shorter construct encoding a shorter Gag fusion may allow for higher incorporation of PEmax.

[0019] Example 5 shows prime editing constructs coupled to the envelope protein VSV-G, incorporation into VLPs and targeted gene editing in cells exposed to such VLPs. This approach may allow one protein (like PEmax) to be fused to VSV-G, whereas another protein may be fused to Gag or GagPro. It is furthermore demonstrated that the method leads to higher level of prime editing than the GagPro-PEmax configuration and provide similar levels of editing as VLPs produced with PH- supported GagPro-PEmax or PH-supported Gag-PEmax recruitment to the membrane. Example 6 shows that functional VLPs can be produced comprising VSV-G fusion proteins devoid of structural viral proteins (Gag / GagPol), which supports targeted gene editing in cells exposed to such particles. This approach may allow a nuclease (like PEmax) to be fused to VSV-G. The production of EVs without GagPol provides similar levels of editing as VLPs but without the need of including and transferring the viral core including the Gag polypeptide.

[0020] Example 7 shows that inclusion of nuclear export signal (NES) in GagPro configuration (PH-GagPro-PEmax) supports increased prime editing rates in LVNP- treated cells.

[0021] Example 8 shows that incorporation of a nicking guide RIMA (ngRNA) in LVNPs leads to improved prime editing in LVNP-treated cells.

[0022] Thus, an object of the present invention relates to the provision of improved protein incorporation of a prime editing system into VLPs. Non-limiting examples of improvements are:

[0023] High gene editing in cells exposed to the VLPs.

[0024] - Surprising high efficiency in spite of the large size of the prime editing system.

[0025] Ribonucleoprotein incorporation into VLPs possible by fusing to both Gag, GagPro (Example 4) and GagPol (Example 2).

[0026] Ribonucleoprotein incorporation into VLPs possible by fusing to VSV-G (example 5).

[0027] PH domain fusion improves prime editing by VLPs (example 3).

[0028] Functional VLPs comprising VSV-G fusion protein can be produced in the absence of Gag and Pol (example 6).

[0029] - VSV-G fusions surprisingly show lower levels of degradation than Gag / GagPro fusions (example 5).

[0030] In particular, it is an object of the present invention to provide a prime editing system that solves the above-mentioned problems of the prior art with delivery efficiency and modification efficiency. Thus, one aspect of the invention relates to a virus-like particle (VLP) comprising a prime editing (PE) system, wherein the nuclease, such as Cas9n, of the PE system is fused to

[0031] - a viral envelope protein, such as VSV-G; and / or

[0032] - a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide.

[0033] In a related aspect the invention relates to a virus-like particle (VLP) comprising a prime editing (PE) system,

[0034] The VLP comprises

[0035] - a viral envelope protein, such as VSV-G; the prime editing system comprises

[0036] - a Cas9n nuclease fused to a reverse transcriptase;

[0037] - a prime editor guide RIMA (pegRNA); wherein the Cas9n nuclease is fused to the viral envelope protein, such as VSV-G; and / or

[0038] - the Cas9n nuclease is fused to a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N-terminal end of the Gag containing polypeptide. wherein the fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit; and wherein the Gag containing polypeptide is HIV derived, preferably HIV-1 derived.

[0039] Another aspect of the present invention relates to a packaging system for producing a VLP according to the invention, the packaging system comprising

[0040] - a packaging plasmid coding for a Gag containing polypeptide and / or a viral envelope protein fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; - an expression plasmid coding for a prime editor guide RIMA (pegRNA) under the control of a promoter;

[0041] - optionally, a packaging plasmid coding for a Gagpol, such as GagPol (D64V);

[0042] - optionally, a packaging plasmid coding for a VSV-G, preferably wt-VSV-G; and

[0043] - optionally, a packaging plasmid coding for a Rev.

[0044] Yet another aspect of the present invention is to provide a method of producing a VLP according to the invention, said method comprising the steps of a) providing packaging system according to the invention; b) transfecting said packaging system into a producer cell; and c) purifying VLPs from said producer cell.

[0045] Still another aspect of the present invention is to provide a fusion protein comprising

[0046] - a viral envelope protein, such as VSV-G fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE; or

[0047] - a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE.

[0048] An aspect of the invention also relates to a nucleic acid, such as a plasmid, coding for a fusion protein according to the invention, preferably under the control of a promoter enabling expression in an expression system.

[0049] Yet an aspect relates to a ribonucleoprotein comprising

[0050] - the fusion protein according to the invention; and

[0051] - a prime editor guide RNA (pegRNA).

[0052] Still an aspect relates to the In vitro use of the VLP according the invention and / or a VLP produced by a method according the invention, and / or a composition according the invention for genome engineering or cell engineering. Yet another aspect relates to the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention, for use as a medicament.

[0053] Brief description of the figures

[0054] Figure 1

[0055] Figure 1 shows (top) Prime editing is initiated with the binding of the PE / pegRNA complex to its genomic target DNA. The SpCas9(H840A) nickase then nicks the PAM-containing strand, following which the 3' end of the nicked DNA strand hybridizes with the complementary primer binding site (PBS) of the pegRNA, allowing the RT to use the genomic DNA strand itself as a primer for reverse transcription. The desired edit is then reverse transcribed from the RT template (RTT) of the pegRNA and incorporated directly into the genomic DNA strand, (bottom) Following reverse transcription by the PE / pegRNA complex, the edited DNA strand is copied onto the non-edited strand through DNA repair by the cell. To bias repair towards the non-edited strand, the 3rd and 5th generation of prime editors (PE3 and PE5, respectively) use an auxiliary nicking sgRNA to generate a nick in the non-edited strand. In the 4th (PE4) and 5th generation of prime editors a dominant negative hMLHl protein is also transiently expressed to inhibit mismatch repair (MMR), further increasing editing efficacies.

[0056] Figure 2

[0057] Figure 2 shows that PE-VLPs can be manipulated to package PEmax / epegRNA RNPs. This can be done either by fusing the PEmax to VSV-G, or as shown in this illustration, by fusing it to the C-terminal protease (Pro) protein. To increase packaging efficiency, a Pleckstrin homology domain (PH) may furthermore be fused to the C-terminal matrix (MA) protein. Packaging of the epegRNA is accomplished by overexpressing the epegRNA in the producer cells, which results in the formation of PEmax / epegRNA ribonucleoprotein complexes (RNPs). By including a protease recognition site between the PEmax and Pro protein, the PEmax / epegRNA RNPs is released in mature viral particles and delivered to the nucleus of the target cell by two possible mechanisms: Transport of the RNPs within the viral core or diffusion through the cytoplasm. Figure 3

[0058] Figure 3 shows vector schematics of different prime editing-fusion plasmids.

[0059] Figure 4

[0060] Figure 4 shows vector schematics of common PE-VLP production plasmids. For production of VLPs containing PE / epegRNA RNPs, one of the constructs shown in Figure 3 may be added to the cocktail of plasmids shown in figure 4.

[0061] Figure 5

[0062] Figure 5 shows plasmid ratios for production of different PE-VLP preparations.

[0063] Figure 6

[0064] Figure 6 shows gene disruption measured by Indel rates following administration of Cas9 incorporating VLPs.

[0065] Figure 7

[0066] Figure 7 shows the percentage of intended editing (correct edit) and unwanted editing (indels) in the HEK3 locus 3 days after transduction of HEK293T cells with GagPro-PEmax and GagProPEmaxARH VLPs (500 ng p24).

[0067] Figure 8

[0068] Figure 8 shows the percentage of PE-protein detected by western blot in VLPs. GagPro-PEmax, GagPro-PEmaxARH, and PH-GagPro-PEmax.

[0069] Figure 9

[0070] Figure 9 shows the percentage of intended editing (correct edit) in the HEK3 locus 3 days after transduction of HEK293T cells with GagProPEmax and PH-GagPro- PEmax VLPs. Figure 10

[0071] Figure 10 shows the percentage of intended editing (correct edit) in the GATA2 gene 3 days after transduction of HEK293T cells with PHGagPro-PEmax VLPs.

[0072] Figure 11

[0073] Figure 11 shows the percentage of intended editing (correct edit) in the HEK3 locus 3 days after transduction of HEK293T cells with GagProPEmax and PH-Gag- PEmax VLPs.

[0074] Figure 12

[0075] Figure 12 shows the percentage of intended editing (correct edit) in the GATA2 gene 3 days after transduction of HEK293T cells with PHGag-PEmax VLPs.

[0076] Figure 13

[0077] Figure 13 shows Western blot of VSV-G-Cas9-VLPs produced with different plasmid production ratios. Cas9 detection was done using an existing FLAG tag.

[0078] Figure 14

[0079] Figure 14 shows P24-normalized dose response with VSV-G-Cas9-VLPs. Detection of Cas9 activity by d2eGFP knockout three days post transduction, in HeLa cells. The ratio denotes the relationship between VSV-G:VSV-G-Cas9 plasmid during VLP production.

[0080] Figure 15

[0081] Figure 15 shows CTT-insertion (correct edit) in the HEK3 locus by VSV-G-PEmax in HEK293T cells three days post transduction. Quantified by next generation sequencing.

[0082] Figure 16

[0083] Figure shows 16 shows Cas9-normalized dose response with VSVG-Cas9 EVs and VLPs. Detection of Cas9 activity by knockout of AFF1 three days post transduction, in HEK293T cells. Figure 17

[0084] Figure 17 shows improved LVNP-directed prime editing leading to a CTT insertion in the HEK3 locus by inclusion of 3xNES in GagPro construct and by co-packaging of nicking guide RNAs (ngRNAs). (A) Schematic representation of the different transfer plasmids used for production. (B) Correct CTT insertion in HEK293T cells determined with Sanger sequencing 72 hours after treatment in a dose-response assay with the indicated LVNPs. All datapoints represent the mean of biological triplicates.

[0085] The present invention will now be described in more detail in the following.

[0086] Detailed description of the invention

[0087] Definitions

[0088] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0089] Fusion gene

[0090] In the present context, the term "fusion gene" refers to a hybrid gene formed from two or more previously separated genes.

[0091] Fusion protein

[0092] In the present context, the term "fusion protein" refers to a hybrid protein formed from combination of two or more proteins.

[0093] CRISPR / Cas9

[0094] In the present context, the term "CRISPR / Cas9" refers to an RNA-guided targeted genome editing tool allowing e.g. for gene knockout, knock-in, insertions and deletions in cell lines and animals. The CRISPR / Cas9 genome editing system requires two components, Cas9, the RNA-guided endonuclease, and a guide RNA (gRNA); the gRNA guides Cas9 to the location in the genome sequence specifically through base pairing between the gRNA and the targeted DNA sequence. Targeted binding of the Cas9 / gRNA complex leads to formation of a double-stranded break in the DNA at a position strictly dictated by interaction between gRNA and target DNA. Prime editing

[0095] Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. For the present case "Prime Editing", "prime editing" and "PE" may be used interchangeably. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RIMA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

[0096] Prime editing involves two major components:

[0097] - A prime editing guide RNA (pegRNA), capable of

[0098] I. identifying the target nucleotide sequence to be edited, and II. encoding new genetic information that replaces the targeted sequence.

[0099] The pegRNA consists of an extended single guide RNA containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During genome editing, the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.

[0100] It is to be understood that that the pegRNA comprises the function of a single guide RNA (sgRNA) and contains the "spacer" sequence that directs the Cas9 H840A nickase portion of the fusion protein to nick the non-edited DNA strand. If not otherwise stated, this sgRNA function is considered part of the pegRNA function. This spacer normally positioned in the 5'-end of the pegRNA.

[0101] - A fusion protein consisting of a nuclease supporting prime editing, such as a Cas9 nickase, fused to a reverse transcriptase. Cas9 H840A nickase

[0102] The introduction of a H840A substitution in Cas9, through which the 840° amino 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. In an embodiment of the invention, the nickase is SpCas9 H840A nickase.

[0103] M-MLV reverse transcriptase:

[0104] M-MLV reverse transcriptase (M-MLV RT) is an enzyme that synthesizes DNA from a single-stranded RNA template. In an embodiment of the invention, the reverse transcriptase is an M-MLV reverse transcriptase.

[0105] Indels

[0106] In the present context, the term "indels" refers to insertion-deletion mutations and is possibly formed when repair of the DNA is performed by non-homologous end-joining (NHEJ). These are likely to disrupt the reading frame if the cut site is located within the coding region of a gene.

[0107] Gag and GacjPol

[0108] As known in the art, Gag and GagPol 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. Gag is composed of at least three protein units: matrix protein (MA), capsid protein (CA) and nucleocapsid protein (NC), whereas Pol consists of retroviral protease (PR), reverse transcriptase (RT) and integrase (IN).

[0109] Preferably, the Gag protein according to the invention is derived from Lentivirus, such as HIV derived, preferably HIV-1 derived. In the example section Gag / Pol protein from HIV-1 has been tested.

[0110] GagPro

[0111] In the present context "GagPro" refers to a truncated version GagPol lacking both reverse transcriptase (RT) and integrase (IN). GagPro still contains the Gag and protease (Pro) unit, and is referred to as GagPro in here. Preferably, the GagPro protein according to the invention is derived from Lentivirus such as HIV derived, preferably HIV-1 derived. In the example section Gag protein from HIV-1 has been tested. sgRNA

[0112] In the present context, the term "sgRNA" refers to a single guide RIMA, which enables highly efficient and accurate editing. "gRNA" is used interchangeably with "sgRNA". The sgRNA comprises two parts - a targeting region and a non-targeting region. The sgRNA may further comprise a variable number of uracil nucleotides at the 3'end. The number of uracil nucleotides depend on expression system used.

[0113] In one embodiment, said non-targeting region is downstream of said targeting region. Hereby, is to be understood that the non-targeting region is positioned at the 3' end of the targeting region.

[0114] It should be understood that the "targeting sequence" relates to the part of the sgRNAs, which is complementary to the "target site" in the genome. The exact length of the targeting region in the sgRNAs may vary. Thus, in one embodiment, the targeting region has a length of 17-24 nucleotides, such as 18-22, like 19-21, such as 20 nucleotides.

[0115] Sequence identity

[0116] In the present context, the term "sequence identity" is here defined as the sequence identity between genes or proteins at the nucleotide, base or amino acid level, respectively. Specifically, a DNA and an RNA sequence are considered identical if the transcript of the DNA sequence can be transcribed to the identical RNA sequence.

[0117] Thus, in the present context "sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes {e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences ( / .e., % identity = # of identical positions / total # of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length.

[0118] In another embodiment, the two sequences are of different length and gaps are seen as different positions. One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention.

[0119] To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilizing the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http: / / www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov / cgi-bin / BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous. The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation.

[0120] Base pair

[0121] In the present context, the term "base pair" refers to two nucleotides being arranged in a secondary structure in a way enabling the two nucleotides to perform a base pairing.

[0122] Base pairing

[0123] In the present context, when referring to a "base pairing", G pairs to C, A pairs to T and U and vice versa. In some embodiments, G may also pair to U and vice versa to form a so-called wobble base pair. A wobble base pair is a non-Watson- Crick base pairing between two nucleotides in RIMA molecules. The four main wobble base pairs are guanine-uracil, inosine-uracil, inosine-adenine, and inosinecytosine (G-U, I-U, I-A and I-C).

[0124] Substitutions

[0125] In the present context, the term "substitutions" refers to exchange of one nucleotide with a different nucleotide.

[0126] Modifications

[0127] In the present context, the term "modifications" refers to the change of a sequence by substitution of nucleotide(s), deletion of nucleotide(s) and / or insertion of nucleotide(s).

[0128] Extension

[0129] In the present context, the term "extension" refers to the elongation of a secondary structure of the sgRNA.

[0130] Knockout

[0131] In the present context, the term "knockout" refers to the removal of a gene, part of a gene or disruption of the reading frame for the gene whereby the gene is no longer correctly transcribed or translated. Donor or donor sequence

[0132] In the present context, the terms "donor" or "donor sequence" refer to a DNA sequence to be introduced into the genome at the double-stranded cut performed by the CRISPR / Cas complex. A donor sequence contains two stretches of DNA. In one embodiment, the donor sequence contains two stretches of DNA with homology to regions flanking the genomic cut site. Administration of the donor sequence allows the cut sit to be repaired by homologous recombination.

[0133] Homology arms

[0134] In the present context, the term "homology arms" refers to a left (LHA) and right (RHA) arranged on either side of the donor sequence to be inserted. The homology arms are designed to flank the Cas9 cleavage site.

[0135] A virus-like particle CVLP)

[0136] In the present context, the terms "virus-like particle" or "VLP", refer to particles containing a lipid membrane comprising one or more virus-derived components, such as Gag, GagPol and / or VSV-G.

[0137] In an embodiment, the VLP may be free from lentivirus components or even be free from Gag and Pro protein and only comprise VSV-G and VSV-G fused to the PE system.

[0138] If the one or more virus-derived components are derived from lentivirus, the VLP may be named "Lentivirus-derived particle", "Lentivirus-derived NanoParticle" or "LVNP". In the present context, VLP and LVNP may be used interchangeably.

[0139] Lentivirus-derived particle / Lentivirus-derived NanoParticle / LVNP

[0140] In the present context, the terms "lentivirus-derived particle", "Lentivirus-derived NanoParticle" and "LVNP" are used interchangeably. "LVNP" is short for Lentivirus- derived NanoParticle. LVNP is a virus-like particle derived from lentivirus. The LVNP is capable of introducing proteins and nucleic acids / nucleotides to a cell by transduction but is not capable of integrating and replicating itself in the host.

[0141] In one embodiment, the LVNP would comprise features from the lentivirus, which enables it to deliver proteins and nucleic acids / nucleotides to the cell such as the Gag polyprotein resulting in Matrix, Capsid and Nucleocapsid proteins and the protease from the Pol polyprotein. VSV-G

[0142] The envelope protein of vesicular stomatitis virus G glycoprotein (VSV-G). A commonly used envelope protein for pseudotyping is VSV-G. VSV-G is a trimeric protein that binds phosphatidylserine and low-density lipoprotein receptors (LDLRs) on a cell surface to endocytose into the cell. VSV-G is commonly used as it is good at infecting most cells.

[0143] Pleckstrin homology domain (PH domain)

[0144] Pleckstrin homology domain (PH domain) or (PHIP) or simply "PH" is a protein domain of approximately 120 amino acids that occurs in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton. PH domains for instance play a role in recruiting proteins to different membranes, thus targeting them to appropriate cellular compartments.

[0145] Vector system or packaging system

[0146] In the present context, the term "vector system" or "packaging system" refer to a system comprising packaging plasmids and potentially one or more transfer / expression plasmid(s). The packaging plasmids encode the necessary components for vector production (including the fusion protein of the invention) and the transfer / expression plasmid carries gene(s) of interest such as pegRNA.

[0147] Virus-like particle (VLP)

[0148] As described above and also outlined in the example section, in here is disclosed improved fusion strategies for loading of prime editing systems into VLPs. Thus, an aspect of the invention relates to a virus-like particle (VLP) comprising a prime editing (PE) system, wherein the nuclease, such as Cas9n, of the PE system is fused to

[0149] - a viral envelope protein, such as VSV-G; and / or

[0150] - a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide.

[0151] In a related aspect the invention relates to a virus-like particle (VLP) comprising a prime editing (PE) system, The VLP comprises a viral envelope protein, such as VSV-G; the prime editing system comprises

[0152] - a Cas9n nuclease fused to a reverse transcriptase;

[0153] - a prime editor guide RIMA (pegRNA); wherein the Cas9n nuclease is fused to the viral envelope protein, such as VSV-G; and / or

[0154] - the Cas9n nuclease is fused to a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N-terminal end of the Gag containing polypeptide. wherein the fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit; and wherein the Gag containing polypeptide, is HIV derived, preferably HIV-1 derived.

[0155] In an embodiment, the nuclease of the PE system is fused to the viral envelope protein VSV-G, preferably the nuclease is fused to the C-terminal of the viral envelope protein via the N-terminal of nuclease of the ribonucleoprotein. Example 5 shows prime editing constructs coupled to the envelope protein VSV-G, incorporation into VLPs and targeted gene editing in cells exposed to such VLPs. This approach may allow one protein (like PEmax) to be fused to VSV-G, whereas another protein may be fused to Gag or GagPro. It is furthermore demonstrated that the method leads to higher level of prime editing than the GagPro-PEmax configuration and provide similar levels of editing as VLPs produced with PH- supported GagPro-PEmax or PH-supported Gag-PEmax recruitment to the membrane. Lower degradation of these fusions also appears to be a result of this fusion strategy.

[0156] Example 6 shows that the Gag and / or GagPro protein may be completely avoided, when fusing to the envelope protein, such as VSV-G. In an embodiment, the viral envelope glycoprotein is an adenoviral envelope glycoprotein, an adeno-associated viral envelope glycoprotein, a retroviral envelope glycoprotein, or a lentiviral envelope glycoprotein.

[0157] In a preferred embodiment, the viral envelope glycoprotein is a retroviral envelope glycoprotein.

[0158] In yet an embodiment, the viral envelope glycoprotein is a vesicular stomatitis virus G protein (VSV-G), a baboon retroviral envelope glycoprotein (BaEVRIess), a measles virus glycoprotein, a nipah virus glycoprotein, a SARS-Cov-2 spike protein, a FuG-B2 envelope glycoprotein, an HIV-1 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein.

[0159] In a preferred embodiment, the viral envelope (glycol)protein is VSV-G protein. In the example section (see e.g. examples 5 and 6) fusions to VSV-G have been tested.

[0160] In another embodiment, the nuclease of the PE system is fused to a Gag containing polypeptide selected from the group consisting of Gag, GagPol, GagPro, preferably the nuclease is fused to the C-terminal of the Gag containing polypeptide via the N-terminal of the nuclease of the PE system. Example 3 demonstrates that addition of a Pleckstrin homology (PH) domain to the GagPro- PEmax configuration supports production of VLPs showing higher levels of prime editing in recipient cells. These findings suggest that the capacity of the PH domain to bind phosphatidylinositol in the plasma membrane supports better recruitment of the GagPro-PEmax polypeptide allowing potentially more PH- GagPro-PEmax polypeptides to be packaged in each VLP unit.

[0161] Example 4 shows effective prime editing in cells treated with VLPs packaged with PH-Gag-PEmax polypeptides. This demonstrates that protease derived from normal GagPol polypeptides, which are co-incorporated into the VLPs during production, can act on PH-Gag-PEmax and support release PEmax / pegRNA RNPs during VLP maturation after budding. Thus, a shorter construct encoding a shorter Gag fusion may allow for higher packaging. In an embodiment, the Gag containing polypeptide is encapsulated by a lipid membrane and a viral envelope glycoprotein, such as VSV-G protein.

[0162] In another embodiment, the fusion is through a linker, preferably a peptide linker.

[0163] In a related embodiment, the linker comprises a reverse transcriptase. Thus, the linker may contain the reverse transcriptase of the PE system. The linker may also comprise Pro.

[0164] In a preferred embodiment, the linker comprises or consists of one or more elements selected from the group consisting of a PCS (protease cleavage site), a FLAG-tag, such as lxFLAG, 2xFLAG and 3xFLAG, an NLS (nuclear localization signal), an NES (nuclear export sequence), a self-cleaving peptide, such as a 2A self-cleaving peptide, such as T2A, P2A, E2A, and F2A, and combinations thereof, preferably the linker comprises a PCS a FLAG-tag and an NLS. In the example section linkers comprising e.g. a PCS a FLAG-tag and an NLS have been tested.

[0165] In a preferred embodiment, the linker comprises a NES, such as selected from SEQ ID NO: 19-21, or a 3xNES, such as SEQ ID NO: 22. Example 7 shows the improved effect of incorporating a 3xNES (SEQ ID NO: 22).

[0166] Thus, in an embodiment, a protease cleavage site (PCS) is positioned between the Gag containing polypeptide or the viral envelope protein and the nuclease, such as Cas9n, of the PE system, preferably the PCS is SQNYPIVQ.

[0167] The VLP according to the invention may comprise additional proteins or be free of certain proteins. Thus, in an embodiment, the VLP

[0168] I. comprises unfused VSV-G (not fused to the nuclease of the PE system), such as wt VSV-G; and / or

[0169] II. comprises unfused VSV-G (not fused to the nuclease of the PE system) and unfused Gag (not fused to the nuclease of the PE system); and / or

[0170] III. comprises unfused VSV-G (not fused to the nuclease of the PE system) and GagPol, such as GagPol(D64V); and / or

[0171] IV. comprises Rev. As shown in the example section it is preferred that the VLP also comprises wt VSV-G (unfused to a PE system) and / or free (unfused to a PE system) Gag, GagPol or GagPol(D64V). It is also shown that the addition of Rev may not be required.

[0172] In an embodiment, the VLP

[0173] V. is free of Rev; and / or

[0174] VI. is free of unfused Pol (not fused to the nuclease of the PE system); and / or

[0175] VII. is free of unfused Gag (not fused to the nuclease of the PE system) and / or

[0176] VIII. is free of unfused GagPol (not fused to the nuclease of the PE system).

[0177] As shown in the example section, the VLP may be free of Rev. Example 6 surprisingly shows that addition of unfused Pol, Gag or GagPol is not required for producing functional VLPs.

[0178] The prime editing (PE) system is well known in the art. Thus, in an embodiment, the PE system comprises

[0179] - a nuclease supporting prime editing, preferably a nickase, such as Cas9n or SpCas9n, more preferably SpCas9-H840A, fused to a reverse transcriptase (RT), and

[0180] - a prime editor guide RNA (pegRNA).

[0181] Figure 1 outlines a specific embodiment of the PE system.

[0182] The molar ratio between the two main components of the PE system may influence the efficiency. Thus, in an embodiment, the molar ratio between the nuclease supporting prime editing and the pegRNA in the VLP is 2: 1-1:2, such as around 1: 1.

[0183] In an embodiment, the pegRNA

[0184] I. identifies the target nucleotide sequence to be edited, and

[0185] II. encodes for new genetic information that replaces the targeted sequence. In another embodiment, during genome editing, the primer binding site of the pegRNA allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.

[0186] Optimized PE systems have been developed. Thus, in an embodiment, the pegRNA is an engineered pegRNA (epegRNA).

[0187] In a related embodiment, the epegRNA is selected from the group consisting of epegRNA comprising one, two or more 3'-pseudoknots, such as modified prequeosinei-1 riboswitch aptamer, tevopreQl, and the truncated frameshifting pseudoknot from Moloney murine leukemia virus (MMLV) (tmpknot).

[0188] Different types of reverse transcriptases have been optimized for PE. Thus, in an embodiment, the reverse transcriptase is selected from the group consisting of wildtype Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, M-MLV RT(D200N / L603W / T330P / T306K / W313F), and MMLV reverse transcriptase comprises a C-terminal amino acid truncation to remove the endogenous MMLV protease cleavage site. In an embodiment, the C-terminal amino acid truncation is about 1-180, about 1-170, about 1-160, about 1-150, about 1-140, about 1-130, about 1-120, about 1-110, about 1-100, about 1-90, about 1-80, about 1-70, about 1-60, about 1-50, about 1-40, about 1- 30, about 1-20, or about 1-10 amino acids in length. Modified reverse transcriptases are used in the PE2 system.

[0189] In an embodiment, the PE system further comprises a second sgRNA complementary to the genomic DNA in proximity to the cleavage / nicking site of the nuclease, such as 0-150 nucleotides, preferably, 0-100, more preferably the nicking guide (ngRNA) is only complementary to the genomic DNA after gene editing by the PE system. Such second sgRNAs are used in the PE3 and PE5 systems. Thus, in an embodiment, the PE system further comprises a ngRNA, such as SEQ ID NO: 23. Example 8 shows the improved effect of incorporating ngRNAs in the VLPs, namely SEQ ID NO: 23.

[0190] In an embodiment, the PE system further comprises a dominant negative MLH1.

[0191] Dominant negative MLH1 is used in the PE4 and PE5 systems. Thus, in an embodiment, the prime editor comprises PE2, PE3, PE4, PE5, PE2max, PE3max, PE4max, or PE5max.

[0192] Different types of nucleases can be used in PE systems. Thus, in an embodiment, the nuclease, supporting prime editing is a Cas9, preferably a Cas9 nickase, more preferably selected from the group consisting of SpCas9-H840A, SpCas9-D10A, a SaCas9 nickase, SpG nickase and SpRY Cas9 nickase. In the example section SpCas9-H840A has been used.

[0193] In an embodiment, the Cas9 protein is a Cas9 protein supporting nuclease prime editing.

[0194] As outlined above and in the example section, it has been found that the VLPs according to the present invention support incorporation of the large PE system. Thus, in an embodiment, the nuclease part of the PE system has a size of at least 190 KDa, preferably such as at least 220 KDa, such as at least 250 KDa, or such as in the range 190 KDa to 400 KDa, such as in the range 190-300 KDa, such as in the range 190-270 KDa.

[0195] In a related embodiment, the ribonuclease part of the PE system has a size of at least 217 KDa, preferably such as at least 247 KDa, such as at least 277 KDa, or such as in the range 217 KDa to 427 KDa, such as in the range 217-327 KDa, such as in the range 217-297 KDa.

[0196] Different types of VSV-G may be used for the fusion proteins. Thus, in an embodiment, VSV-G is selected from the group consisting of wt VSV-G, VSV-G variants; S162T, T230N, T368A, K66T, T368A, and / or E380K and truncated versions of VSV-G such as 21-amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED). Wt VSV-G can be found e.g. in Addgene#12259 (pMD.2G). VSV-G variants are e.g. described in [PMID:23364315] . Truncated versions are e.g. described in [PMID: 16267571] .

[0197] In an embodiment, fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit. In another embodiment, the Gag containing polypeptide or the viral envelope protein and the nuclease, such as Cas9n, of the PE system has been transcribed and translated as a single unit.

[0198] In yet another embodiment, the nuclease, such as Cas9n, of the PE system supporting prime editing and the reverse transcriptase (RT) has been transcribed and translated as a single unit.

[0199] In yet another embodiment, the Gag containing polypeptide or the viral envelope protein and the nuclease, such as Cas9n, of the PE system and the reverse transcriptase (RT) has been transcribed and translated as a single unit.

[0200] As outlined in the example section (see e.g. example 3), a PH domain may improve the efficacy of the VLPs. Thus, in an embodiment, the VLP according to the invention, comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of Gag containing polypeptide or the viral envelope protein.

[0201] In a related embodiment, the Pleckstrin homology (PH) domain is positioned at the N-terminal end of Gag and / or N-terminal end of GagPro or the N-terminal end of VSV-G, preferably at the N-terminal end of the fusion protein comprising the nuclease, such as Cas9n, of the PE system.

[0202] Compositions

[0203] The VLPs according to the invention may form part of a composition. Thus, an aspect of the invention relates to a composition comprising the VLP according to the invention or a plurality of VLPs according to the invention.

[0204] The composition may also be a pharmaceutical composition. Thus, a further aspect of the invention relates to a pharmaceutical composition comprising the VLP according to the invention.

[0205] In an embodiment, the pharmaceutical composition further comprises a pharmaceutical acceptable carrier, enhancer and / or diluent. Packaging system

[0206] The VLPs according to the invention may be produced from a packing system. Thus, an aspect of the invention relates to a packaging system for producing a VLP according to the invention, the packaging system comprising

[0207] - a packaging plasmid coding for a viral envelope protein fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; AND / OR a packaging plasmid coding for a Gag containing polypeptide fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain;

[0208] - an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;

[0209] - optionally, a packaging plasmid coding for a Gagpol, such as GagPol (D64V);

[0210] - optionally, a packaging plasmid coding for a VSV-G, preferably wt-VSV-G; and

[0211] - optionally, a packaging plasmid coding for a Rev.

[0212] Thus, the packaging system comprises a plasmid encoding fusion proteins (fused to the nuclease part of the PE system), plasmids encoding pegRNA and optionally other plasmid encoding proteins supporting correct packaging and delivery, such as GagPol (unfused to PE parts), (unfused to PE parts) VSV-G and Rev. Such different packaging systems have been tested in the example section.

[0213] In an embodiment, a packaging plasmid encodes for a viral envelope protein, preferably VSV-G, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter.

[0214] In an embodiment, the viral envelope protein is selected from the group consisting of wt VSV-G, VSV-G variants; S162T, T230N, T368A, K66T, T368A, and / or E380K and truncated versions of VSV-G such as 21-amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED) and VSV- G-PH, wherein the PH domain is fused to the cytoplasmic tail of VSV-G. In an embodiment, a packaging plasmid encodes for a Gag containing polypeptide, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain.

[0215] In an embodiment, the Gag containing fusion polypeptide is selected from the group consisting of PH-GagPol, PH-GagPro, and PH-Gag.

[0216] In a preferred embodiment, the packaging system comprises

[0217] - a packaging plasmid encoding for unfused Gag and / or GagPol under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or

[0218] - a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or

[0219] - a packaging plasmid encoding for Rev under the control of a promoter.

[0220] As shown in the example section, it may be advantageous to use additional packaging plasmids.

[0221] In another preferred embodiment, the packaging system comprises

[0222] - a packaging plasmid coding for a viral envelope protein, preferably VSV-G, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter;

[0223] - an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;

[0224] - a packaging plasmid coding for unfused Gag and / or GagPol viral genes under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or

[0225] - a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or

[0226] - optionally, a packaging plasmid encoding for Rev viral gene under the control of a promoter.

[0227] For example, in example 5 VLPs are produced using such a packaging system. In another preferred embodiment, the packaging system comprises

[0228] - a packaging plasmid coding for a Gag containing polypeptide and / or a viral envelope protein fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain;

[0229] - an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;

[0230] - a packaging plasmid coding for unfused Gag and / or GagPol viral genes under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or

[0231] - a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or

[0232] - optionally a packaging plasmid encoding for Rev viral gene under the control of a promoter.

[0233] For example, in example 3 VLPs are produced using such a packaging system.

[0234] In yet another preferred embodiment, the packaging system comprises

[0235] - a packaging plasmid coding for a viral envelope protein, preferably VSV-G, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter;

[0236] - an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;

[0237] - optionally, a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or wherein the packaging system is free from expression plasmids or constructs coding for unfused Gag and / or GagPol viral genes under the control of a promoter. In Example 6, VLPs are produced using such a packaging system.

[0238] In an embodiment, the packaging system comprises an expression plasmid coding for a nicking guide RNA (ngRNA) under the control of a promoter. Such a system is illustrated in figure 17A. Methods of preparing VLP

[0239] The present invention also relates to a method of producing the VLPs according to the invention. Thus, an aspect of the invention relates to a method of producing a VLP according to the invention, said method comprising the steps of a) providing packaging system according to the invention; b) transfecting said packaging system into a producer cell; and c) purifying VLPs from said producer cell.

[0240] In an embodiment, the molar ratio between the plasmid encoding viral envelope protein, preferably VSV-G fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE to the plasmid encoding unfused VSV-G is 95:5 to 5:95 such as 90: 10 to 10:90, such as 80:20 to 20:80, like 70:30 to 30:70, such as 60:40 to 40:60, like 50:50, such as 40:60 to 60:40.

[0241] In another embodiment the molar ratio between the plasmid coding for a Gag containing polypeptide fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE to the plasmid coding for unfused Gagpol, preferably D64V, is 95:5 to 5:95 such as 90: 10 to 10:90, such as 80:20 to 20:80, like 70:30 to 30:70, such as 60:40 to 40:60, like 50:50, such as 40:60 to 60:40.

[0242] In an embodiment, said producer cell is a mammalian producer cell line, preferably a human producer cell line.

[0243] In yet an embodiment, said producer cell is selected from the group consisting of HEK293T cells, such as Lenti-X / 293T cells, COS-1 cells, K562 cells, and AML12 cells. In the example section HEK293T cells have been used.

[0244] VLP obtained by or obtainable by the method of the invention.

[0245] An aspect of the invention also relates to a VLP obtained by or obtainable by a method according to the invention. Fusion protein

[0246] The present invention also relates to fusion proteins suitable for prime editing. Thus, an aspect of the invention relates to a fusion protein comprising

[0247] - a viral envelope protein, such as VSV-G fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE; or

[0248] - a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE.

[0249] In the example section such fusion proteins have been tested in VLPs comprising a PE system.

[0250] In an embodiment, the fusion protein comprises the viral envelope protein VSV-G or a truncated version thereof, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE.

[0251] In another embodiment, the fusion protein comprises a Gag containing polypeptide selected from the group consisting of PH-Gag, PH-GagPol and PH- GagPro.

[0252] In an embodiment, the Gag containing polypeptide or viral envelope protein is fused to the nuclease supporting PE, through a linker, preferably a peptide linker. Different suitable linkers are disclosed in the section relating to VLPs.

[0253] In a preferred embodiment, the fusion protein comprises the structure:

[0254] [VSV-G]-[cleavable Linker]-[nuclease supporting PE]-[Reverse transcriptase]; or

[0255] [VSV-G]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase];

[0256] [PH domain]-[Gag]-[cleavable Linker]-[nuclease supporting PE]-[Reverse transcriptase]; or [PH domain]-[Gag]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE] -

[0257] [Reverse transcriptase]; or

[0258] [PH domain]-[GagPol]-[cleavable Linker]-[nuclease supporting PE]-

[0259] [Reverse transcriptase]; or

[0260] [PH domain]-[GagPol]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase];

[0261] [PH domain]-[GagPro]-[cleavable Linker]-[nuclease supporting PE] -

[0262] [Reverse transcriptase]; or

[0263] [PH domain]-[GagPro]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase] .

[0264] The structures above, have all been tested in the example section.

[0265] In alternative embodiments, the fusion protein is a structure selected from the group consisting of:

[0266] [nuclease supporting PE]-[Reverse transcriptase]-[cleavable Linker]-[PH domain]-[Gag];

[0267] [FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase]-

[0268] [PCS]-[PH domain]-[Gag];

[0269] [nuclease supporting PE]-[Reverse transcriptase]-[cleavable Linker]-[PH domain]-[GagPol];

[0270] [FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase]-

[0271] [PCS]-[PH domain]-[GagPol];

[0272] [nuclease supporting PE]-[Reverse transcriptase]-[cleavable Linker]-[PH domain]-[GagPro];

[0273] [FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase]-

[0274] [PCS]-[PH domain]-[GagPro];

[0275] [PH domain]-[GagPol]-[cleavable Linker]-[nuclease supporting PE]- [Reverse transcriptase];

[0276] [PH domain]-[GagPol]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE]-[Reverse transcriptase];

[0277] [PH domain]-[cleavable linker]-[nuclease supporting PE]-[Reverse transcriptase] -[cleavable linker] -[Gag Pol];

[0278] [PH domain]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE] - [Reverse transcriptase] - [ NLS] -[PCS] - [Gag Pol]; [PH domain]-[cleavable linker]-[nuclease supporting PE]-[Reverse transcriptase] -[cleavable linker] -[Gag Pro];

[0279] [PH domain]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE] - [Reverse transcriptase] - [ NLS] -[PCS] - [Gag Pro];

[0280] [PH domain]-[cleavable linker]-[nuclease supporting PE]-[Reverse transcriptase]-[cleavable linker] -[Gag] ; and

[0281] [PH domain]-[PCS]-[FLAG-tag(s)]-[NLS]-[nuclease supporting PE] - [Reverse transcriptase] -[NLS] -[PCS] -[Gag].

[0282] In an embodiment, the fusion protein is a structure selected from the group consisting of:

[0283] [PH domai n] -[Gag Pro] -[3xNES]- [PCS] -[FLAG-tag(s)]-[NLS(s)]-[ nuclease supporting PE]-[NLS(s)]-[Reverse transcriptase]-[NLS(s)]; and

[0284] [PH domai n] - [Gag] -[3xNES]- [PCS] -[FLAG-tag(s)]-[NLS(s)]- [nuclease supporting PE]-[NLS(s)]-[Reverse transcriptase]-[NLS(s)];

[0285] It is to be understood that embodiments relating to the fusion proteins according to the invention are also combinable with the other aspect of the invention, such as the VLPs.

[0286] Nucleic acid

[0287] The fusion proteins according to the invention may be encoded from a nucleic acid construct. Thus, an aspect of the invention relates to a nucleic acid, such as a plasmid, coding for a fusion protein according to the invention, preferably under the control of a promoter enabling expression in an expression system.

[0288] Ribonucleoprotein

[0289] The fusion protein according to the invention may also for part of a ribonucleoprotein together with a pegRNA. Thus, yet an aspect of the invention relates to a ribonucleoprotein comprising

[0290] - the fusion protein according to the invention; and

[0291] - a prime editor guide RNA (pegRNA).

[0292] In an embodiment, the ribonucleoprotein further comprises a ngRNA. The skilled person can design pegRNA for a desired target using his common general knowledge.

[0293] Cells

[0294] The VLP, the fusion protein, the nucleic acid and the ribonucleoprotein according to the invention are, during production, present inside a (producer) cell line. Thus, an aspect of the invention relates to a cell comprising the VLP, and / or fusion protein and / or the nucleic acid and / or the ribonucleoprotein according to the invention.

[0295] In vitro use

[0296] In an aspect, the invention relates to the in vitro use of the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention for genome engineering or cell engineering.

[0297] In an embodiment, the genome engineering or cell engineering is in an eukaryotic cell, such as a yeast cell, a mammalian cell, such as a human cell.

[0298] In an embodiment, the cells corrected by said VLP has a percentage of correct edits of at least 20%, such as at least 40%, such as least 50%, such as at least 60%, like at least 70%, such as at least 80%, like at least 90%, such as at least 95%, like at least 98%, such as at least 99%.

[0299] Medical uses

[0300] The VLPs according to the invention can of course also be used for medical purposes. The skilled person can design pegRNAs for any desired target and use it according to the present invention as a medicament. Hence, a further aspect of the present invention relates to the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention, for use as a medicament.

[0301] In a related aspect the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention for use in the treatment or alleviation of a disorder treatable by prime editing.

[0302] In an embodiment, the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention for use in the treatment or alleviation of disorder selected from the group consisting of monogenic diseases, such as, sickle cell disease, beta thalassemia, inborn errors of immunity (GATA2-deficiency (MonoMac), chronic- granulomatous disease, X-linked chronic granulomatous disease, hyper IgM syndrome, hyper IgE syndrome), cystic fibrosis, hemophilia, Wilson's disease, glycogen storage disease, hereditary angioedema, Duschenne muscular dystrophy, myotonic dystrophy type 1.

[0303] In an embodiment, the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention for use in the treatment or alleviation of disorder selected from the group consisting of diseases of the eye, such as age-related macular degeneration, Leber's congenital amaurosis, and retinitis pigmentosa.

[0304] Additional aspects of the invention

[0305] In an aspect the invention relates to a method for treating or alleviating a condition or disease in a subject in need thereof, the method comprising administering to the subject the VLP according to the invention and / or a VLP produced by a method according to the invention, and / or a composition according to the invention.

[0306] In an embodiment, the subject suffers from a disease or disorder treatable by prime editing.

[0307] Items of the inventions

[0308] 1. A virus-like particle (VLP) comprising a prime editing (PE) system, wherein the nuclease, such as Cas9n, of the PE system is fused to

[0309] - a viral envelope protein, such as VSV-G; and / or a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide.

[0310] 2. The VLP according to item 1, wherein the nuclease of the PE system is fused to the viral envelope protein VSV-G, preferably the nuclease is fused to the C- terminal of the viral envelope protein via the N-terminal of nuclease of the ribonucleoprotein.

[0311] 3. The VLP according to item 1, wherein the nuclease of the PE system is fused to a Gag containing polypeptide selected from the group consisting of Gag, GagPol, GagPro, preferably the nuclease is fused to the C-terminal of the Gag containing polypeptide via the N-terminal of the nuclease of the PE system.

[0312] 4. The VLP according to any of the preceding items, wherein the Gag containing polypeptide is encapsulated by a lipid membrane and a viral envelope glycoprotein, such as VSV-G protein.

[0313] 5. The VLP according to any of the preceding items, wherein the fusion is through a linker, preferably a peptide linker.

[0314] 6. The VLP according to any of the preceding items, wherein the linker comprises a reverse transcriptase.

[0315] 7. The VLP according to item 4 or 5, wherein the linker comprises or consists of one or more elements selected from the group consisting of a PCS, a FLAG-tag, such as lxFLAG, 2xFLAG and 3xFLAG, an NLS, an NES, a self-cleaving peptide, such as a 2A self-cleaving peptide, such as T2A, P2A, E2A, and F2A, and combinations thereof, preferably the linker comprises a PCS a FLAG-tag and an NLS.

[0316] 8. The VLP according to any of the preceding items

[0317] I. comprising unfused VSV-G (not fused to the nuclease of the PE system), such as wt VSV-G; and / or II. comprising unfused VSV-G (not fused to the nuclease of the PE system) and unfused Gag (not fused to the nuclease of the PE system); and / or

[0318] III. comprising unfused VSV-G (not fused to the nuclease of the PE system) and GagPol, such as GagPol(D64V); and / or

[0319] IV. comprising Rev; and / or

[0320] V. being free of Rev; and / or or

[0321] VI. being free of unfused Pol (not fused to the nuclease of the PE system); and / or

[0322] VII. being free of unfused Gag (not fused to the nuclease of the PE system) and / or

[0323] VIII. being free of unfused GagPol (not fused to the nuclease of the PE system).

[0324] 9. The VLP according to any of the preceding items, wherein the PE system comprises

[0325] - a nuclease supporting prime editing, preferably a nickase, such as Cas9n or SpCas9n, more preferably SpCas9-H840A, fused to a reverse transcriptase (RT), and

[0326] - a prime editor guide RNA (pegRNA).

[0327] 10. The VLP according to any of the preceding items, wherein, in the VLP the molar ratio between the nuclease supporting prime editing and the pegRNA is 2: 1- 1 :2, such as around 1 : 1.

[0328] 11. The VLP according to item 9 or 10, wherein the pegRNA

[0329] I. identifies the target nucleotide sequence to be edited, and

[0330] II. encodes for new genetic information that replaces the targeted sequence.

[0331] 12. The VLP according any of items 9-11, wherein the pegRNA is an engineered pegRNA (epegRNA). 13. The VLP according to item 12, wherein the epegRNA is selected from the group consisting of epegRNA comprising one, two or more 3'-pseudoknots, such as modified prequeosinei-1 riboswitch aptamer, tevopreQl, and the truncated frameshifting pseudoknot from Moloney murine leukemia virus (MMLV) (tmpknot).

[0332] 14. The VLP according to any of items 9-13, wherein during genome editing, the primer binding site of the pegRNA allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.

[0333] 15. The VLP according to any of items 9-14, wherein the reverse transcriptase is selected from the group consisting of wildtype Moloney Murine Leukemia Virus (M- MLV) reverse transcriptase, and M-MLV RT(D200N / L603W / T330P / T306K / W313F)).

[0334] 16. The VLP according to any of the preceding items, wherein the PE system further comprises a second sgRNA complementary to the genomic DNA in proximity to the cleavage / nicking site of the nuclease, such as 0-150 nucleotides, preferably, 0-100, more preferably the nicking guide (ngRNA) is only complementary to the genomic DNA after gene editing by the PE system, such as the ngRNA being SEQ ID NO: 23.

[0335] 17. The VLP according to any of the preceding items, wherein the PE system according to any of the preceding items, further comprising a dominant negative MLH1.

[0336] 18. The VLP according to any of the preceding items, wherein the nuclease, supporting prime editing is a Cas9, preferably a Cas9 nickase, more preferably selected from the group consisting of SpCas9-H840A, SpCas9-D10A, a SaCas9 nickase, SpG nickase and SpRY Cas9 nickase.

[0337] 19. The VLP according to any of the preceding items 9-18, wherein the Cas9 protein is a Cas9 protein supporting nuclease prime editing.

[0338] 20. The VLP according to any of the preceding items, wherein the nuclease part of the PE system has a size of at least 190 KDa, preferably such as at least 220 KDa, such as at least 250 KDa, or such as in the range 190 KDa to 400 KDa, such as in the range 190-300 KDa, such as in the range 190-270 KDa.

[0339] 21. The VLP according to any of the preceding items, wherein the ribonuclease part of the PE system has a size of at least 217 KDa, preferably such as at least 247 KDa, such as at least 277 KDa, or such as in the range 217 KDa to 427 KDa, such as in the range 217-327 KDa, such as in the range 217-297 KDa.

[0340] 22. The VLP according to any of the preceding items, wherein the VSV-G is selected from the group consisting of wt VSV-G, VSV-G variants; S162T, T230N, T368A, K66T, T368A, E380K and truncated versions of VSV-G such as 21-amino- acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED).

[0341] 23. The VLP according to any of the preceding items, wherein a protease cleavage site (PCS) is positioned between the Gag containing polypeptide or the viral envelope protein and the nuclease, such as Cas9n, of the PE system, preferably the PCS is SQNYPIVQ.

[0342] 24. The VLP according to any of the preceding items, wherein fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit.

[0343] 25. The VLP according to any of the preceding items, wherein the Gag containing polypeptide or the viral envelope protein and the nuclease, such as Cas9n, of the PE system has been transcribed and translated as a single unit.

[0344] 26. The VLP according to any of the preceding items, wherein the nuclease, such as Cas9n, of the PE system supporting prime editing and the reverse transcriptase (RT) has been transcribed and translated as a single unit.

[0345] 27. The VLP according to any of the preceding items, wherein the Gag containing polypeptide or the viral envelope protein and the nuclease, such as Cas9n, of the PE system and the reverse transcriptase (RT) has been transcribed and translated as a single unit. 28. The VLP according to any of the preceding items, comprising a Pleckstrin homology (PH) domain, preferably at the N-terminal end of Gag containing polypeptide or the viral envelope protein.

[0346] 29. The VLP according to item 28, wherein the Pleckstrin homology (PH) domain is positioned at the N-terminal end of Gag and / or N-terminal end of GagPro or the N-terminal end of VSV-G, preferably at the N-terminal end of the fusion protein comprising the nuclease, such as Cas9n, of the PE system.

[0347] 30. A composition comprising the VLP according to any of the preceding items or a plurality of VLPs according to any of the preceding items.

[0348] 31. A pharmaceutical composition comprising the VLP according to any of item 1- 29.

[0349] 32. The pharmaceutical composition according to item 31, further comprising a pharmaceutical acceptable carrier, enhancer and / or diluent.

[0350] 33. A packaging system for producing a VLP according to any of the preceding items 1-29, the packaging system comprising

[0351] - a packaging plasmid coding for a viral envelope protein fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; AND / OR a packaging plasmid coding for a Gag containing polypeptide fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain;

[0352] - an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;

[0353] - optionally, a packaging plasmid coding for a Gagpol, such as GagPol (D64V);

[0354] - optionally, a packaging plasmid coding for a VSV-G, preferably wt-VSV-G; and

[0355] - optionally, a packaging plasmid coding for a Rev. 34. The packaging system according to item 33, wherein a packaging plasmid encodes for a viral envelope protein, preferably VSV-G, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter.

[0356] 35. The packaging system according to item 33 or 34, wherein the viral envelope protein is selected from the group consisting of wt VSV-G, VSV-G variants; S162T, T230N, T368A, K66T, T368A, E380K and truncated versions of VSV-G such as 21- amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV- G (VSV-GED) and VSV-G-PH, wherein the PH domain is fused to the cytoplasmic tail of VSV-G.

[0357] 36. The packaging system according to item 33, wherein a packaging plasmid encodes for a Gag containing polypeptide, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter.

[0358] 37. The packaging system according to item 36, wherein the Gag containing fusion polypeptide is selected from the group consisting of PH-GagPro, PH-GagPol and PH-Gag.

[0359] 38. The packaging system according to any of items 33-37, comprising

[0360] - a packaging plasmid encoding for unfused Gag and / or GagPol under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or

[0361] - a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or

[0362] - a packaging plasmid encoding for Rev under the control of a promoter.

[0363] 39. The packaging system according to any of items 33-38, comprising

[0364] - a packaging plasmid coding for a viral envelope protein fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; AND / OR a packaging plasmid coding for a Gag containing polypeptide and / or a viral envelope protein fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE, under the control of a promoter; wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain;

[0365] - an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;

[0366] - a packaging plasmid coding for unfused Gag and / or GagPol viral genes under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or

[0367] - a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or

[0368] - a packaging plasmid encoding for Rev viral gene under the control of a promoter.

[0369] 40. A method of producing a VLP according to any of the preceding items 1-29, said method comprising the steps of a) providing packaging system according to any of items 33-39; b) transfecting said packaging system into a producer cell; and c) purifying VLPs from said producer cell.

[0370] 41. The method according to item 40, wherein the ratio between the plasmid encoding viral envelope protein, preferably VSV-G fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE to the plasmid encoding unfused VSV-G is 95:5 to 5:95 such as 90: 10 to 10:90, such as 80:20 to 20:80, like 70:30 to 30:70, such as 60:40 to 40:60, like 50:50, such as 40:60 to 60:40.

[0371] 42. The method according to item 40, wherein the ratio between the plasmid coding for a Gag containing polypeptide fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE to the plasmid coding for unfused Gagpol, preferably D64V, is 95:5 to 5:95 such as 90: 10 to 10:90, such as 80:20 to 20:80, like 70:30 to 30:70, such as 60:40 to 40:60, like 50:50, such as 40:60 to 60:40. 43. The method according to any of items 40-42, wherein said producer cell is a mammalian producer cell line, preferably a human producer cell line.

[0372] 44. The method according to any of items 40-43, wherein said producer cell is selected from the group consisting of HEK293T cells, such as Lenti-X / 293T cells, COS-1 cells, K562 cells, and AML12 cells.

[0373] 45. A VLP obtained by or obtainable by a method according to any of items 40-44.

[0374] 46. A fusion protein comprising

[0375] - a viral envelope protein, such as VSV-G fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE; or

[0376] - a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE.

[0377] 47. The fusion protein according to item 46, wherein the Gag containing polypeptide is selected from the group consisting of PH-Gag, PH-GagPro, and PH- GagPol.

[0378] 48. The fusion protein according to item 46, comprising the viral envelope protein VSV-G or a truncated version thereof, fused to a nuclease supporting PE, such as Cas9n, fused to a reverse transcriptase (RT) supporting PE.

[0379] 49. The fusion protein according to any of the preceding items 46-48, wherein the Gag containing polypeptide or the viral envelope protein is fused to the nuclease supporting PE, such as Cas9n, through a linker, preferably a peptide linker.

[0380] 50. A nucleic acid, such as a plasmid, coding for a fusion protein according to any of items 46-49, preferably under the control of a promoter enabling expression in an expression system.

[0381] 51. A ribonucleoprotein comprising the fusion protein according to any of items 46-49; and a prime editor guide RIMA (pegRNA).

[0382] 52. In vitro use of the VLP according to any of items 1-29 or 45 and / or a VLP produced by a method according to any of items 40-44, and / or a composition as described in any of items 30-32 for genome engineering or cell engineering.

[0383] 53. The in vitro use according to item 52, wherein the genome engineering or cell engineering is in a eukaryotic cell, such as a yeast cell, a mammalian cell, such as a human cell.

[0384] 54. The in vitro use according to item 52 or 53, wherein cells corrected by said VLP has a percentage of correct edits of at least 20%, such as at least 40%, such as least 50%, such as at least 60%, like at least 70%, such as at least 80%, like at least 90%, such as at least 95%, like at least 98%, such as at least 99%.

[0385] 55. The VLP according to any of items according to any of items 1-29 or 45 and / or a VLP produced by a method according to any of items 40-44, and / or a composition as described in any of items 30-32, for use as a medicament.

[0386] 56. The VLP according to any of items according to any of items 1-29 or 45 and / or a VLP produced by a method according to any of items 40-44, and / or a composition as described in any of items 30-32 for use in the treatment or alleviation of a disorder treatable by prime editing.

[0387] 57. The VLP according to any of items 1-29 or 45 and / or a VLP produced by a method according to any of items 40-44, and / or a composition as described in any of items 30-32 for use in the treatment or alleviation of a disorder selected from the group consisting of monogenic diseases, such as, sickle cell disease, beta thalassemia, inborn errors of immunity (GATA2-deficiency (MonoMac), chronic- granulomatous disease, X-linked chronic granulomatous disease, hyper IgM syndrome, hyper IgE syndrome), cystic fibrosis, hemophilia, Wilson's disease, glycogen storage disease, hereditary angioedema, Duschenne muscular dystrophy, myotonic dystrophy type 1. 58. The VLP according to any of items according to any of items 1-29 or 45 and / or a VLP produced by a method according to any of items 40-44, and / or a composition as described in any of items 30-32 for use in the treatment or alleviation of a disorder selected from the group consisting of diseases of the eye, such as age-related macular degeneration, Leber's congenital amaurosis, and retinitis pigmentosa.

[0388] 59. A method for treating or alleviating an condition or disease in a subject in need thereof, the method comprising administering to the subject the VLP according to any of items 1-29 or 45 and / or a VLP produced by a method according to any of items 40-44, and / or a composition as described in any of items 30-32.

[0389] 60. The method according to item 59, wherein the subject suffers from a disease or disorder treatable by prime editing.

[0390] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0391] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0392] The invention will now be described in further details in the following non-limiting examples.

[0393] Examples

[0394] Example 1 - Production of engineered VLPs carrying the prime editor (PE) protein complexed with prime editing guide RNA (pegRNA)

[0395] Aim of study

[0396] In our early studies, which showed incorporation of transposase, ZFNs, TALENs, and Cas9 into VLPs, we used strategies that fused the foreign nonviral protein to the N-terminal end of Gag / GagPol or to the C-terminal end of GagPol. Although these strategies worked well for packaging of shorter proteins into VLPs, we set out here to establish other fusion strategies that could potentially compensate for the larger size of the PE protein.

[0397] Materials and Methods

[0398] We established four fusion strategies for packaging of PE / pegRNA RNPs into VLPs. An overview of these four configurations, referred to as GagPro-PEmax, PH- GagPro-PEmax, PH-Gag-PEmax, and VSV-G-PEmax, are provided in Figure 3. The examples below demonstrate the use of each of these strategies to deliver PE in VLPs. The materials and Methods related to the production and functional analyses of VLPs apply to all four configurations, and are described here in Example 1. Figure 4 shows the cocktail of plasmids used for a typical VLP production. Figure 5 provides an overview of the amounts / ratios of the different plasmids used for the different configurations and mark the specific differences between the VLP production schemes.

[0399] Cell lines

[0400] All VLP productions were carried out in LentiX / HEK293T cells. Cells treated with VLPs to test PE / pegRNA transfer leading to prime editing included HEK293T cells and K562 cells. All cells were cultured in DMEM (Sigma-Aldrich) supplemented with 5% FBS, 100 U / ml penicillin, and 100 pg / ml streptomycin (Thermo Fischer Scientific). Cells were maintained at 60-90% confluence at 37°C with 5% carbon dioxide and tested negative for mycoplasma (Eurofins Genomics).

[0401] Plasmid construction

[0402] Plasmids were constructed using NEBuilder® HiFi DNA Assembly Master Mix (New England BioLabs). In brief, the plasmid backbone was linearized by digestion with the relevant restriction enzyme (Thermo Fischer Scientific) and insert(s) were PCR amplified from plasmid DNA and / or synthetic DNA (TWIST Bioscience). The resulting fragments were combined using NEBuilder® HiFi DNA Assembly Master Mix (New England BioLabs) according to manufacturer's instructions. All sgRNAs were designed using Synthego software or CRISPOR13and pegRNAs by PegIT14(sequences are provided in the Sequences section below).

[0403] VLP production VLPs were produced by transient transfection of Lenti-X (Takara Bio). Cells were seeded at a density of 3.75-4.00 x 106in 10-cm dishes and incubated overnight. A calcium phosphate solution15containing the relevant plasmid cocktail (Figures 3-5) was dropwise added to 10-cm dishes and incubated overnight. The medium was replenished after 24h, and the supernatant was harvested 48 hours and 72 hours post-transfection. The supernatant was passed through a 0.45 um filter and concentrated 300x by ultracentrifugation in a cushion of 20% (w / v) sucrose and PBS unless otherwise stated. Ultracentrifugation was performed at 25,000 RPM at 4°C for 2 hours in a Beckman SW27 or SW28 rotor. Pellets were resuspended in 85 pL PBS overnight (4°C), pooled (first and second harvest), and centrifuged at 1,200 RPM to pellet residual debris. The VLP concentration was quantified by p24 ELISA (XpressBio) according to the manufacturer's protocol and stored at -80C in aliquots until use.

[0404] Detection of PE in VLP by Western blotting

[0405] For detection of PE in VLPs, ultracentrifuged particles (90 ng p24) were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with 10 mM NaF and 1 x complete protease inhibitor cocktail (Roche). The lysate was denatured in XT Sample Buffer supplemented with XT Reducing Agent (Bio-Rad), separated by SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk dissolved in TBS / 0.05% Tween-20 for 1 hour and incubated overnight with a FLAG antibody (Sigma-Aldrich). The membrane was washed and incubated with anti-mouse secondary antibodies (Dako) and visualized by chemiluminescence using Clarity Western ECL Substrate (Bio-Rad). The antibodies were removed with stripping buffer (Thermo Fisher Scientific), and the membrane was incubated overnight with a p24 antibody (R&D Systems) followed by anti-rabbit secondary antibodies.

[0406] Treatment of recipient cells with VLP

[0407] Cells were plated in 24-well plates (Sarstedt) at a density of 5 x 104cells / well (HEK293T) and incubated overnight. A mastermix containing the indicated amount of VLP (p24 normalized) and DMEM containing polybrene (8 pg / mL) was prepared separately for each condition. For the 24-well format, 500 pL mastermix was applied to replica wells and incubated overnight. Cells were harvested 3 days post-transduction and used for downstream analysis. Genomic DNA extraction and PCR for detection of edits

[0408] Genomic DNA was isolated by NaCI / EtOH precipitation as previously described8. Following resuspension in TE-buffer or ddFhO, lpL (1-20 ng) was used for PCR amplification of the target region using Phusion Master Mix (Thermo Fisher Scientific). PCR products were purified by gel extraction (Omega Bio-tek) or PCR clean-up (SAP / EXO). A solution of 0.5 pL FastAP, 0.5 pL Exol, 9 pL PCR product, and H2O to a final volume of 18 pL was incubated at 37°C for 15 min followed by inactivation at 85°C for 15 min in a thermocycler. The resulting amplicon was sequenced by Eurofins Genomics. The resulting indel frequencies were deconvoluted by ICE analysis16or DECODR17and CRISPessov218for prime editing.

[0409] Example 2 - Incorporation of PEmax in VLPs by fusion to C-terminus of truncated GagPro (GagPro-PEmax) leads to prime editing in recipient cells

[0410] Aim of study

[0411] In previous VLP protein incorporation strategies, we fused small endonucleases to the N-terminus of Gag / GagPol (Data not shown). We also used this strategy to package PEmax / pegRNA in VLPs, but observed very low levels of prime editing in cells treated with such VLPs (data not shown). Therefore, to accommodate the large PE protein (plus the pegRNA), we wanted to study PE incorporation in VLPs by fusing the protein to the C-terminus of a truncated version GagPol lacking both reverse transcriptase and integrase. Since this poplypeptide still contained the protease (Pro) unit, we refer to this configuration as GagPro-PEmax.

[0412] Materials and Methods

[0413] Experiments were carried out according to details provided in Example 1.

[0414] Results

[0415] To further improve VLP-based editing rates, we engineered particles that could potentially better accommodate large fusion proteins. We deleted regions encoding reverse transcriptase and integrase from the pol gene, allowing production of a polypeptide containing Gag, the viral protease (Pro), and the protein of interest (POI), the latter fused to the C-terminus of Gag through a linker containing a PCS, 3xFLAG, and an additional NLS to enhance nuclear localization19. This configuration, referred to as GagPro-POI, was validated by fusing SpCas9 to the C-terminus of GagPro. By packaging GagPro-SpCas9 polypeptides in the VLPs, we found slightly improved levels of gene disruption of two loci, Fah and Pcsk9, as compared to a configuration where SpCas9 was fused to the N-terminus of Gag (Figure 6).

[0416] To investigate the capacity of VLPs to accommodate and deliver prime editors, we constructed VLPs carrying GagPro-PEmax polypeptides (PEmax described in20). Also, in a separate preparation of VLPs, we included a GagPro polypeptide fused to a truncated version of PEmax, PEmaxARH, which contains an MLV-RT domain devoid of the RNase H domain21(Figure 3).

[0417] To support RNP stability, we expressed and incorporated engineered pegRNAs (epegRNAs) containing a structural motif (tevopreQl) at the 3' end to minimize degradation22. By exposing HEK293T cells to VLPs loaded with PEmax and an epegRNA targeting HEK3 (CTTins), we found robust levels of CTT insertion (6%) in the HEK3 locus without detectable indel formation (<0.01%) using targeted next-generation sequencing (Figure 7). For PEmaxARH, we found comparable editing rates (5%) without indel formation (Figure 7).

[0418] Conclusion

[0419] Collectively, these data demonstrate the capacity of VLPs to deliver larger genome editing toolkits including prime editing RNPs consisting of PEmax (with a size of 230 kDa) complexed with epegRNA. These findings may pave the way for the development of VLP-based formulations for therapeutic delivery of prime editors allowing gene editing without DSB formation and the need of an HDR donor sequence.

[0420] Example 3 - Inclusion of Pleckstrin homology domain in GagPro configuration (PH-GagPro-PEmax) supports increased prime editing rates in VLP-treated cells

[0421] Aim of study

[0422] To further improve the capacity of VLPs to support prime editing, we aimed at optimizing the recruitment of the GagPro-PEmax polypeptide to the membrane during VLP production. For this purpose, we studied the impact of adding a Pleckstrin homology (PH) domain to the GagPro-PEmax polypeptide (see example 2).

[0423] Materials and Methods

[0424] Experiments were carried as described in Example 1.

[0425] Results

[0426] First, we produced VLPs with PH-GagPro-PEmax configuration and with the GagPro-PEmax as control. For both VLP preparations, we detected incorporation of the PE protein in the particles (Figure 8). We targeted the HEK3 locus in HEK293T cells and treated the cells with two different dosages of VLP (corresponding to 250 ng and 500 ng p24). The intended edit sequence (a CTT insertion) was detected with both configurations and with both dosages (Figure 9). Using GagPro-PEmax, VLP delivery supported insertion of the correct edit sequence in up to 10% of the alleles in the population supporting our previous findings (Example 2). However, using PH-GagPro-PEmax for production, treatment with VLPs resulted in up 20% editing.

[0427] Next, we developed pegRNA for the GATA2 locus and produced PH-GagPro-PEmax VLPs for this particular. Again, using this configuration, we measured high levels of prime editing resulting in insertion of the correct edit in up to 25% of the alleles with a VLP dose corresponding to 500 ng p24 (Figure 10).

[0428] Conclusion

[0429] Our data demonstrate that addition of a Pleckstrin homology (PH) domain to the GagPro-PEmax configuration supports production of VLPs showing higher levels of prime editing in recipient cells. These findings suggest that the capacity of the PH domain to bind phosphatidylinositol in the plasma membrane supports better recruitment of the GagPro-PEmax polypeptide allowing potentially more PH- GagPro-PEmax polypeptides to be packaged in each VLP unit.

[0430] Example 4 - Incorporation of PEmax in VLPs by fusion to C-terminus of Gag (Gag-PEmax) leads to prime editing in recipient cells

[0431] Aim of study To further truncate the polypeptide, we wanted to investigate whether a direct fusion of PEmax to Gag (without Pro) would support PEmax incorporation in VLPs and release after VLP budding and maturation.

[0432] Materials and Methods

[0433] VLP production and functional tests were performed as described in Example 1.

[0434] Results

[0435] To study whether direct fusion of PEmax to Gag supported PEmax incorporation in VLPs and activity in recipient cells, the Pro domain was omitted from the PH-Gag- PEmax. Notably, as shown in Figure 4, VLPs were also in this case produced as a mixture of the normal Gag / GagPol polypeptides and the polypeptide carrying the PEmax fusion domain. This allowed protease (Pro) to be cleaved off the normal GagPol and release PEmax from Gag. We compared the editing capacity of VLPs produced with PH-Gag-PEmax to VLPs produced with GagPro-PEmax. Again, higher levels of editing were observed with PH-containing Gag resulting in insertion of the correct edit (CTT insertion) in up to 15% of the HEK3 alleles in the HEK293T cell population (Figure 11). Overall, since PH-Gag-PEmax supported editing rates that were similar to the rates of PH-GagPro-PEmax (Figure 9), the lack of the Pro domain did not appear to affect the editing efficacy.

[0436] Using the PH-Gag-PEmax configuration for VLP production, we verified potent prime editing in the GATA2 locus in HEK293T cells, resulting in insertion of the desired prime edit in up to 30% of the GATA2 alleles (Figure 12).

[0437] Conclusion

[0438] This example shows effective prime editing in cells treated with VLPs packaged with PH-Gag-PEmax polypeptides. This demonstrates that protease derived from normal GagPol polypeptides, which are co-incorporated into the VLPs during production, can act on PH-Gag-PEmax and support release PEmax / pegRNA RNPs during VLP maturation after budding. Example 5 - Fusion of PEmax to the intracellular domain of the VSV-G envelope protein supports packaging of PEmax in VLPs and high prime editing rates in recipient cells

[0439] Aim of study

[0440] As an alternative approach for packaging PEmax complexed to pegRNA into VLPs, we wanted to test whether endonucleases (like Cas9) and nickases (like PEmax) could be packaged into VLPs through fusion with the VSV-G protein. The aim was to investigate (i) whether the VSV-G envelope protein is capable of supporting incorporation of a large protein fused to the intracellular domain into VLP and (ii) whether VSV-G envelope proteins carrying a terminal fusion domain retains its function to bind receptors on the surface of recipient mediating VLP uptake.

[0441] Materials and Methods

[0442] Incorporation of the protein of interest, e.g., SpCas9 protein, is facilitated by fusing Cas9 or the Cas9-derived effector to the VSV-G glycoprotein, which is expressed in the VLP producer cells and embedded in the envelope surrounding the viral capsid. We originally cloned a plasmid encoding VSV-G fused to spCas9 joined by the SQNY protease cleavage site (PCS), referred to as VPC (for VSVG- PCS-Cas9) (Figure 3). We produced VPC-VLPs by co-transfection of plasmid encoding GagPol(D64V), Rev, VSV-G / VPC and sgRNA. To assess CRISPR / Cas9 activity, we utilized a HeLa / d2eGFP-IRES-Puro reporter cell line. Using a sgRNA directed at d2eGFP, we can quantify the knockout of d2eGFP by flow cytometry, which provides a 1-to-l measure of Cas9 activity at the genomic level. The rapid turnover of d2eGFP allows for efficient separation of positive and negative cells three days after transduction

[0443] Results

[0444] We produced VPC-VLPs (VLP carrying Cas9 fused via a protease cleavage site with VSV-G) using different ratios of VSV-G plasmids and VPC plasmids. To characterize VPC-VLPs and study VSV-G:VPC ratios during production, we performed Western blot to verify the incorporation of Cas9 (Figure 13). In all VPC- VLPs, we detected a Cas9 band at the size corresponding to unfused SpCas9 (163 kDa). This band was not present in the control VLPs produced with 100% VSV-G (100:0). All the production ratios (VSV-G:VPC ratio) resulted in lentiviral particles incorporating Cas9, and potentially the 60:40 ratio incorporated more Cas9 compared to 90: 10. P24 was blotted as a loading control (data not shown). The Western blot indicated that Cas9 was released upon particle maturation. A high- molecular band was present in all VPC-VLPs, suggesting the presence of uncleaved VPC (Cas9 163 kDa+ VSV-G 56 kDa).

[0445] Interestingly, VPC-VLPs (VSV-G fusions) resulted in remarkably clean lanes. In contrast, with Gag-based fusion strategies we have often seen several smaller bands, indicative of potential degradation products, generated by the protease activity during particle maturation (Figure 8).

[0446] We then quantified the number of d2eGFP-positive cells by flow cytometry three days after treatment of d2eGFP reporter cells with P24-normalized dosages of VLP, allowing us to estimate d2eGFP KO rates for the different VLPs preparations (Figure 14). We observed highly efficient, dose-dependent knockout of d2eGFP for all VPC-VLP ratios and no activity of the controls (100:0 ratio). Neither 20 nor 40 ng P24 doses allowed us to discriminate between the ratios based on knockout effectiveness. However, the lowest dose corresponding to 10 ng P24 revealed a difference between the 90: 10 ratio (70.4 ± 0.8% knockout) and the 70:30 ratio (57 ± 1.7% knockout) (Figure 14). Altogether, these findings demonstrated effective Cas9-based DNA cleavage leading to indel formation in cells treated with VLPs carrying VPC fusions.

[0447] To incorporate PEmax by fusion to VSV-G glycoprotein into VLPs, we cloned a plasmid encoding VSV-G fused to PEmax via by the SQNY protease cleavage site (PCS) (Figure 3). We produced VSV-G-PCS-PEmax VLPs by co- transfecting plasmids encoding GagPol(D64V), Rev, VSV-G / VSV-G fusion and epegRNA (for CTT insertion in the HEK3 locus). HEK293T cells treated with 10 and 20 pL VLPs were sequenced by next generation sequencing, demonstrating 20-24% CTT insertion at the HEK3 locus (Figure 15).

[0448] Conclusion

[0449] We show here a unique way to incorporate prime editing tools (PE protein complexed with pegRNA) into VLP, which supports targeted gene editing in cells exposed to such particles. This approach may allow one protein (like PEmax) to be fused to VSV-G, whereas another protein can be fused to Gag or GagPro. We demonstrate that the method leads to higher level of prime editing than the GagPro-PEmax configuration and provide similar levels of editing as VLPs produced with PH-supported GagPro-PEmax or Gag-PEmax recruitment to the membrane.

[0450] Example 6 - Fusion to VSV-G envelope protein supports packaging of nucleases in extracellular vesicles supporting high gene editing rates in recipient cells

[0451] Aim of study

[0452] As an alternative approach for packaging nucleases into VLPs, we wanted to test whether endonucleases (like Cas9) and nickases (like PEmax) could be packaged into extracellular vesicles (EVs) through fusion with the VSV-G protein without the presence of GagPol. The aim was to investigate (i) whether the VSV-G envelope protein is capable of supporting incorporation of a protein fused to the intracellular domain into EVs and (ii) whether VSV-G envelope protein carrying a terminal fusion domain retains its function to bind receptors on the surface of recipient cells mediating EV uptake.

[0453] Materials and Methods

[0454] Incorporation of the protein of interest, e.g., SpCas9 protein, is facilitated by fusing Cas9 or the Cas9-derived effector to the VSV-G glycoprotein, which is expressed in the EV producer cells and embedded in the membrane of budding EVs. We originally cloned a plasmid encoding VSV-G fused to spCas9 joined by the SQNY protease cleavage site (PCS), referred to as VPC (for VSVG-PCS-Cas9) (Figure 14). We produced VPC-VLPs and VPC-EVs based on the ratios listed in Figure 5, however for VPC-EVs, GagPol(D64V) was omitted. This allowed production of VLPs carrying both fusion VSV-G and wildtype VSV-G. To assess CRISPR / Cas9 activity, HEK293 cells were exposed to VSV-G-containing EVs. Using a sgRNA directed at the AFF1 locus, we quantified the nuclease activity at the genomic level by PCR, Sanger sequencing, and indel analysis.

[0455] Results We produced VPC-VLPs and VPC-EVs (both carrying Cas9 fused via a protease cleavage site with VSV-G) by measuring the concentration of Cas9. In both cases, HEK293T cells were exposed to Cas9-normalized preparations pf EV or VLP.

[0456] We observed efficient, dose-dependent knockout of AFF1 for both VPC-VLPs and VPC-EVs. At the maximum dose (30 ng Cas9) VPC-VLPs and VPC-EVs resulted in comparable levels of indel formation. However, at the lowest doses (3.75-15 ng Cas9) VPC-VLPs resulted in higher indel formation (Figure 16). Altogether, these findings demonstrated effective Cas9-based DNA cleavage leading to indel formation in cells treated with EVs carrying the VPC fusion proteins.

[0457] Conclusion

[0458] We show here a unique way to incorporate nucleases into EVs devoid of structural viral proteins (Gag / GagPol), which supports targeted gene editing in cells exposed to such particles. This approach may allow one protein (like PEmax) to be fused to VSV-G. The production of EVs without GagPol provide similar levels of editing as VLPs but without the need of including and transferring the viral core including the Gag polypeptide.

[0459] This strategy is appealing, since (without being bound by theory) such VLPs comprise fewer different virus derived proteins and therefore may have a lower immunogenic effect. Further, patients may show higher tolerance to VLPs containing fewer types of virus proteins.

[0460] Example 7 - Inclusion of nuclear export signal (NES) in GagPro configuration (PH-GagPro-PEmax) supports increased prime editing rates in LVNP-treated cells

[0461] Aim of study

[0462] To facilitate nuclear entry in recipient cells, the PEmax prime editor protein is engineered to contain nuclear localization signals (NLSs). This feature may also drive nuclear entry in LVNP production cells and thus reduce assembly of GagPro polypeptides under the plasma membrane in producer cells. This may lower LVNP yields and potentially performance. To counteract this effect, we aimed at examining the effect of introducing nuclear export signals (NESs) in the GagPro polypeptide. For this purpose, we studied the impact of adding a 3xNES module upstream of the protease cleavage site (PCS) flanking PEmax. This position should allow the 3xNES module to be separated from PEmax during LVNP maturation and therefore not mediate nuclear export of PEmax in LVNP-treated cells.

[0463] Materials and Methods

[0464] Experiments were carried out as described in Example 1. LVNP productions included plasmids are shown in Figure 17A.

[0465] Results

[0466] We produced LVNPs carrying the PH-GagPro-PEmax configuration with and without 3xNES and analyzed the level of prime editing in the HEK3 locus in LVNP- treated HEK293T cells using a dose-escalating approach. The intended edit sequence (a CTT insertion) was detected with both configurations and with all dosages (Figure 17). Without 3xNES (PH-GagPro-PEmax), editing rates ranged from 7 to 20% for increasing dosages of LVNPs, whereas inclusion of the 3xNES module in LVNPs (PH-GagPro-3xNES-PEmax) resulted in editing rates ranging from 7 to 28% (Figure 17B). At all doses (except the lowest dose), LVNPs carrying the PH-GagPro-3xNES-PEmax configuration resulted in higher prime editing levels

[0467] Conclusion

[0468] Our data demonstrate that the addition of nuclear export signal in the PH-GagPro- PEmax configuration supports production of LVNPs showing higher levels of prime editing in recipient cells. This finding suggests that nuclear export of polypeptides during LVNP production supports production of LVNPs with a higher prime editing potential.

[0469] A similar effect is expected for the constructs according to the invention which is free of the Pro domain.

[0470] Example 8 - Incorporation of a nicking guide RNA (ngRNA) in LVNPs leads to improved prime editing in LVNP-treated cells

[0471] Aim of study

[0472] For prime editing, it is known that editing rates are improved by using an auxiliary nicking sgRNAs (ngRNAs) to nick the non-edited strand. To further improve the prime editing rates obtained with LVNP-directed delivery, we investigated whether it was possible to co-pack ngRNAs in the LVNPs together with PEmax and pegRNAs and also any possible effect thereof.

[0473] Materials and Methods

[0474] Experiments were carried out as described in Example 1. LVNP productions included plasmids shown in Figure 17A.

[0475] Results

[0476] We produced LVNPs carrying both pegRNAs (SEQ ID NO: 18) and ngRNAs (SEQ ID NO: 23) by co-transfecting plasmids encoding pegRNAs and ngRNAs together with the remaining plasmids necessary for LVNP production (Figure 17A). We know from our previous observations that incorporation of guide RNAs in LVNP depends on binding to Cas9 or a Cas9-derivative like PEmax. Hence, the rationale was that both pegRNAs and ngRNAs would be included in LVNPs through the interaction with PEmax.

[0477] LVNPs carrying the PH-GagPro-3x-NES-PEmax configuration (SEQ ID NO: 24) were produced with and without inclusion of ngRNA-encoding plasmid, and levels of prime editing in the HEK3 locus were assessed in HEK293T cells. Compared to LVNPs without ngRNAs, LVNPs that also contained ngRNAs induced markedly higher levels of prime editing (Figure 17B). With the highest dose of LVNPs containing ngRNAs, we measured >40% prime editing relative to 28% editing without ngRNAs. At all LVNP doses, except the two lowest doses (3.125 and 6.25 ng p24) ngRNA addition supported a higher level of prime editing.

[0478] Conclusion

[0479] Our data demonstrate that ngRNAs can be packaged together with pegRNAs in LVNPs and support increased levels of prime editing by guiding PEmax protein to nick the unedited strand.

[0480] Summary of data

[0481] Collectively, our data describe a set of fusion protein configurations for production of VLPs engineered to incorporate prime editing PE / pegRNA and PE / ngRNA RNPs. Together, VLPs engineered to package and deliver PE / pegRNA and PE / ngRNA RNPs present a novel strategy for delivery of the prime editing technology with potential clinical application.

[0482] References

[0483] 1 Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229-236, doi: 10.1038 / s41586-020-1978-5 (2020).

[0484] 2 Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823, doi: 10.1126 / science.1231143 (2013).

[0485] 3 Anzalone, A. V. et al. Search-and-replace genome editing without doublestrand breaks or donor DNA. Nature 576, 149-157, doi: 10.1038 / s41586- 019-1711-4 (2019).

[0486] 4 van Haasteren, J., Li, J., Scheideler, O. J., Murthy, N. & Schaffer, D. V. The delivery challenge: fulfilling the promise of therapeutic genome editing. Nat Biotechnol 38, 845-855, doi: 10.1038 / s41587-020-0565-5 (2020).

[0487] 5 Wolff, J. H., Haldrup, J., Thomsen, E. A., Andersen, S. & Mikkelsen, J. G. piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac- Based DNA Transposition. Front Genome Ed 3, 786893, doi: 10.3389 / fgeed.2021.786893 (2021).

[0488] 6 Dever, D. P. et al. CRISPR / Cas9 -globin gene targeting in human haematopoietic stem cells. Nature 539, 384-389, doi: 10.1038 / nature20134 (2016).

[0489] 7 Cai, Y. et al. DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. Nucleic acids research 42, e28, doi: 10.1093 / nar / gktll63gktll63 [pii] (2014).

[0490] 8 Skipper, K. A. et al. Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles. Mol Ther Nucleic Acids 11, 253-262, doi: 10.1016 / j.omtn.2018.02.006 (2018).

[0491] 9 Thomsen, E. A. et al. CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch. Mol Ther Nucleic Acids 29, 563-576, doi: 10.1016 / j.omtn.2022.08.005 (2022).

[0492] 10 Cai, Y, Bak, R. O. & Mikkelsen, J. G. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases. Elife 3, e01911, doi: 10.7554 / eLife.01911 (2014).

[0493] 11 Cai, Y. et al. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors. Elife 5, doi: 10.7554 / eLife.12213 (2016).

[0494] 12 Choi, J. G. et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther 23, 627-633, doi: 10.1038 / gt.2016.27 (2016).

[0495] 13 Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR / Cas9 genome editing experiments and screens. Nucleic Acids Res 46, W242-w245, doi: 10.1093 / nar / gky354 (2018).

[0496] 14 Anderson, M. V., Haldrup, J., Thomsen, E. A., Wolff, J. H. & Mikkelsen, J. G. pegIT - a web-based design tool for prime editing. Nucleic Acids Res 49, W505-w509, doi: 10.1093 / nar / gkab427 (2021).

[0497] 15 Graham, F. L. & van der Eb, A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467, doi: 10.1016 / 0042-6822(73)90341-3 (1973).

[0498] 16 Conant, D. et al. Inference of CRISPR Edits from Sanger Trace Data. Crispr j 5, 123-130, doi: 10.1089 / crispr.2021.0113 (2022). 17 Bloh, K. et al. Deconvolution of Complex DNA Repair (DECODR) : Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger Sequencing Data. Crisprj 4, 120-131, doi: 10.1089 / crispr.2020.0022 (2021).

[0499] 18 Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37 , 224-226, doi : 10.1038 / s41587-019- 0032-3 (2019).

[0500] 19 Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nature Communications 12, 2121, doi: 10.1038 / s41467-021-22295-w (2021).

[0501] 20 Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635-5652. e5629, doi: 10.1016 / j. cell.2021.09.018 (2021).

[0502] 21 Gao, Z. et al. A truncated reverse transcriptase enhances prime editing by split AAV vectors. Mol Ther 30, 2942-2951, doi: 10.1016 / j. ymthe.2022.07.001 (2022).

[0503] 22 Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol 40, 402-410, doi: 10.1038 / s41587-021-01039-7 (2022).

[0504] Table 1: Construct Sequence listing Table 2. DNA Sequences coding for sgRNAs and epegRNAs used in the study tevopreQl RNA motif.

[0505] Table 3 : NES sequences Spacer sequence is highlighted, the remaining is scaffold.

[0506] Table 5: PH-GagPro-3xNES-PEmax sequence

[0507]

[0508] Lowercase and Underlined: PH domain. Uppercase and Italics: Gag. Lowercase and Bold: Pol / Pro. Uppercase and underlined: Overlap of Gag and Pol / Pro. Grayscale: NES. Uppercase and bold: PEmax.

Claims

Claims1. A virus-like particle (VLP) comprising a prime editing (PE) system, The VLP comprises- a viral envelope protein, such as VSV-G; the prime editing system comprises- a Cas9n nuclease fused to a reverse transcriptase;- a prime editor guide RIMA (pegRNA); wherein the Cas9n nuclease is fused to the viral envelope protein, such as VSV-G; and / or- the Cas9n nuclease is fused to a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N-terminal end of the Gag containing polypeptide; wherein the fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit; and wherein the Gag containing polypeptide, is HIV-1 derived.

2. The VLP according to claim 1, wherein the Cas9n nuclease of the PE system is fused to the viral envelope protein VSV-G, preferably the nuclease is fused to the C-terminal of the viral envelope protein via the N-terminal of nuclease of the ribonucleoprotein.

3. The VLP according to claim 1, wherein the Cas9n nuclease of the PE system is fused to the Gag containing polypeptide selected from the group consisting of Gag, GagPol, GagPro, preferably the nuclease is fused to the C-terminal of the Gag containing polypeptide via the N-terminal of the Cas9n nuclease of the PE system.

4. The VLP according to any of the preceding claims, wherein the Gag containing polypeptide is encapsulated by a lipid membrane and a viral envelope glycoprotein, such as VSV-G protein.

5. The VLP according to any of the preceding claims, wherein the fusion is through a linker, preferably a peptide linker.

6. The VLP according to claim 5, wherein the linker comprises a reverse transcriptase.

7. The VLP according to claim 5 or 6, wherein the linker comprises or consists of one or more elements selected from the group consisting of a PCS, a FLAG-tag, such as lxFLAG, 2xFLAG and 3xFLAG, an NLS, an NES, a self-cleaving peptide, such as a 2A self-cleaving peptide, such as T2A, P2A, E2A, and F2A, and combinations thereof, preferably the linker comprises a PCS a FLAG-tag and an NLS.

8. The VLP according to any of claims 5-7, wherein the linker comprises an NES, such as selected from SEQ ID NO: 19-21, or a 3xNES, such as SEQ ID NO:22.

9. The VLP according to any of the preceding claimsI. comprising unfused VSV-G, such as wt VSV-G; and / orII. comprising unfused VSV-G and unfused Gag; and / orIII. comprising unfused VSV-G and GagPol, such as GagPol(D64V); and / orIV. comprising Rev; and / orV. being free of Rev; and / or orVI. being free of unfused Pol; and / orVII. being free of unfused Gag and / orVIII. being free of unfused GagPol.

10. The VLP according to any of the preceding claims, wherein the viral envelope (glycol)protein is an adenoviral envelope glycoprotein, an adeno-associated viral envelope glycoprotein, a retroviral envelope glycoprotein, or a lentiviral envelope glycoprotein; and / or the viral envelope (glycol)protein is a retroviral envelope glycoprotein.and / or the viral envelope glycoprotein is a vesicular stomatitis virus G protein (VSV-G), a baboon retroviral envelope glycoprotein (BaEVRIess), a measles virus glycoprotein, a nipah virus glycoprotein, a SARS-Cov-2 spike protein, a FuG-B2 envelope glycoprotein, an HIV-1 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein; and / or preferably, the viral envelope protein is VSV-G protein.

11. The VLP according to claim 9 or 10, wherein the pegRNAI. identifies the target nucleotide sequence to be edited, andII. encodes for new genetic information that replaces the targeted sequence.

12. The VLP according to any of claims 9-11, wherein the pegRNA is an engineered pegRNA (epegRNA).

13. The VLP according to claim 12, wherein the epegRNA is selected from the group consisting of epegRNA comprising one, two or more 3'-pseudoknots, such as modified prequeosinei-1 riboswitch aptamer, tevopreQl, and the truncated frameshifting pseudoknot from Moloney murine leukemia virus (MMLV) (tmpknot).

14. The VLP according to any of claims 9-13, wherein during genome editing, the primer binding site of the pegRNA allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.

15. The VLP according to any of claims 9-14, wherein the reverse transcriptase is selected from the group consisting of wildtype Moloney Murine Leukemia Virus (M- MLV) reverse transcriptase, and M-MLV RT(D200N / L603W / T330P / T306K / W313F).

16. The VLP according to any of the preceding claims, wherein the PE system further comprises a second sgRNA (nicking guide RIMA (ngRNA)) complementary to the genomic DNA in proximity to the cleavage / nicking site of the nuclease, such as 0-150 nucleotides, preferably, 0-100, more preferably the nicking guide is only complementary to the genomic DNA after gene editing by the PE system.

17. The VLP according to any of the preceding claims, wherein the PE system according to any of the preceding claims, further comprising a dominant negative MLH1.

18. The VLP according to any of the preceding claims, wherein the Cas9 nickase is selected from the group consisting of SpCas9-H840A, SpCas9-D10A, and a SaCas9 nickase.

19. The VLP according to any of the preceding claims, wherein the Cas9 nickase is SpCas9-H840A.

20. The VLP according to any of the preceding claims, wherein the nuclease part of the PE system has a size of at least 190 KDa, preferably such as at least 220 KDa, such as at least 250 KDa, or such as in the range 190 KDa to 400 KDa, such as in the range 190-300 KDa, such as in the range 190-270 KDa.

21. The VLP according to any of the preceding claims, wherein the ribonuclease part of the PE system has a size of at least 217 KDa, preferably such as at least 247 KDa, such as at least 277 KDa, or such as in the range 217 KDa to 427 KDa, such as in the range 217-327 KDa, such as in the range 217-297 KDa.

22. The VLP according to any of the preceding claims, wherein the VSV-G is selected from the group consisting of wt VSV-G, VSV-G variants; S162T, T230N, T368A, K66T, T368A, E380K and truncated versions of VSV-G such as 21-amino- acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED).

23. The VLP according to any of the preceding claims, wherein a protease cleavage site (PCS) is positioned between the Gag containing polypeptide or theviral envelope protein and the Cas9n nuclease, of the PE system, preferably the PCS is SQNYPIVQ.

24. The VLP according to claim 23, wherein the protease cleavage site (PCS) is SQNYPIVQ.

25. The VLP according to any of the preceding claims, wherein the Gag containing polypeptide or the viral envelope protein and the Cas9n nuclease of the PE system has been transcribed and translated as a single unit.

26. The VLP according to any of the preceding claims, wherein the Cas9n nuclease of the PE system and the reverse transcriptase (RT) has been transcribed and translated as a single unit.

27. The VLP according to any of the preceding claims, wherein the Gag containing polypeptide or the viral envelope protein and the Cas9n nuclease of the PE system and the reverse transcriptase (RT) has been transcribed and translated as a single unit.

28. The VLP according to any of the preceding claims, comprising a Pleckstrin homology (PH) domain, preferably at the N-terminal end of Gag containing polypeptide or the viral envelope protein.

29. The VLP according to claim 28, wherein the Pleckstrin homology (PH) domain is positioned at the N-terminal end of Gag and / or N-terminal end of GagPro or the N-terminal end of VSV-G, preferably at the N-terminal end of the fusion protein comprising the Cas9n nuclease of the PE system.

30. A composition comprising the VLP according to any of the preceding claims or a plurality of VLPs according to any of the preceding claims.

31. A pharmaceutical composition comprising the VLP according to any of claim 1- 29.

32. The pharmaceutical composition according to claim 31, further comprising a pharmaceutical acceptable carrier, enhancer and / or diluent.

33. A packaging system for producing a VLP according to any of the preceding claims 1-29, the packaging system comprising- a packaging plasmid coding for a viral envelope protein fused to a Cas9n nuclease, fused to a reverse transcriptase (RT), under the control of a promoter; AND / OR a packaging plasmid coding for a Gag containing polypeptide fused to a Cas9n nuclease , fused to a reverse transcriptase (RT) , under the control of a promoter; wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain;- an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;- optionally, a packaging plasmid coding for a Gagpol, such as GagPol (D64V);- optionally, a packaging plasmid coding for a VSV-G, preferably wt-VSV-G; and- optionally, a packaging plasmid coding for a Rev; wherein the fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit; and wherein the Gag containing polypeptide, is HIV-1 derived.

34. The packaging system according to claim 33, wherein a packaging plasmid encodes for a viral envelope protein, preferably VSV-G, fused to a Cas9n nuclease, fused to a reverse transcriptase (RT), under the control of a promoter.

35. The packaging system according to claim 33 or 34, wherein the viral envelope protein is selected from the group consisting of wt VSV-G, VSV-G variants; S162T, T230N, T368A, K66T, T368A, E380K and truncated versions of VSV-G such as 21- amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV- G (VSV-GED) and VSV-G-PH, wherein the PH domain is fused to the cytoplasmic tail of VSV-G.

36. The packaging system according to claim 33, wherein a packaging plasmid encodes for a Gag containing polypeptide, fused to a Cas9n nuclease, fused to a reverse transcriptase (RT), under the control of a promoter.

37. The packaging system according to claim 36, wherein the Gag containing fusion polypeptide is selected from the group consisting of PH-GagPro, PH-GagPol and PH-Gag.

38. The packaging system according to any of claims 33-37, comprising- a packaging plasmid encoding for unfused Gag and / or GagPol under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or- a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / or- a packaging plasmid encoding for Rev under the control of a promoter.

39. The packaging system according to any of claims 33-38, comprising- a packaging plasmid coding for a viral envelope protein fused to a Cas9n nuclease, fused to a reverse transcriptase (RT), under the control of a promoter; AND / OR a packaging plasmid coding for a Gag containing polypeptide and / or a viral envelope protein fused to a nuclease, such as Cas9n, fused to a reverse transcriptase (RT), under the control of a promoter; wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain;- an expression plasmid coding for a prime editor guide RNA (pegRNA) under the control of a promoter;- a packaging plasmid coding for unfused Gag and / or GagPol viral genes under the control of a promoter, preferably an integrase deficient GagPol, such as GagPol(D64V); and / or- a packaging plasmid encoding for unfused VSV-G viral genes under the control of a promoter, preferably a wt VSV-G; and / ora packaging plasmid encoding for Rev viral gene under the control of a promoter.

40. A method of producing a VLP according to any of the preceding claims 1-29, said method comprising the steps of a) providing packaging system according to any of claims 33-39; b) transfecting said packaging system into a producer cell; and c) purifying VLPs from said producer cell.

41. The method according to claim 40, wherein the ratio between the plasmid encoding viral envelope protein, preferably VSV-G fused to a Cas9n nuclease, fused to a reverse transcriptase (RT) to the plasmid encoding unfused VSV-G is 95:5 to 5:95 such as 90: 10 to 10:90, such as 80:20 to 20:80, like 70:30 to 30:70, such as 60:40 to 40:60, like 50:50, such as 40:60 to 60:40.

42. The method according to claim 40, wherein the ratio between the plasmid coding for a Gag containing polypeptide fused to a Cas9n nuclease, fused to a reverse transcriptase (RT) to the plasmid coding for unfused Gagpol, preferably D64V, is 95:5 to 5:95 such as 90: 10 to 10:90, such as 80:20 to 20:80, like 70:30 to 30:70, such as 60:40 to 40:60, like 50:50, such as 40:60 to 60:40.

43. The method according to any of claims 40-42, wherein said producer cell is a mammalian producer cell line, preferably a human producer cell line.

44. The method according to any of claims 40-43, wherein said producer cell is selected from the group consisting of HEK293T cells, such as Lenti-X / 293T cells, COS-1 cells, K562 cells, and AML12 cells.

45. A VLP obtained by or obtainable by a method according to any of claims 40- 44.

46. A fusion protein comprising- a viral envelope protein, such as VSV-G fused to a Cas9n nuclease, fused to a reverse transcriptase (RT); or- a Gag containing polypeptide, wherein the Gag containing polypeptide further comprises a Pleckstrin homology (PH) domain, preferably at the N- terminal end of the Gag containing polypeptide, fused to a Cas9n nuclease, fused to a reverse transcriptase (RT). wherein the fusions are covalent fusions, such as the proteins being transcribed and translated as a single unit; and wherein the Gag containing polypeptide, is HIV-1 derived.

47. The fusion protein according to claim 46, wherein the Gag containing polypeptide is selected from the group consisting of PH-Gag, PH-GagPro, and PH- GagPol.

48. The fusion protein according to claim 46, comprising the viral envelope protein VSV-G or a truncated version thereof, fused to a Cas9n nuclease, fused to a reverse transcriptase (RT).

49. The fusion protein according to any of the preceding claims 46-48, wherein the Gag containing polypeptide or the viral envelope protein is fused to the Cas9n nuclease, through a linker, preferably a peptide linker.

50. A nucleic acid, such as a plasmid, coding for a fusion protein according to any of claims 46-49, preferably under the control of a promoter enabling expression in an expression system.

51. A ribonucleoprotein comprising- the fusion protein according to any of claims 46-49; and- a prime editor guide RNA (pegRNA).

52. In vitro use of the VLP according to any of claims 1-29 or 45 and / or a VLP produced by a method according to any of claims 40-44, and / or a composition as described in any of claims 30-32 for genome engineering or cell engineering.

53. The in vitro use according to claim 52, wherein the genome engineering or cell engineering is in a eukaryotic cell, such as a yeast cell, a mammalian cell, such as a human cell.

54. The in vitro use according to claim 52 or 53, wherein cells corrected by said VLP has a percentage of correct edits of at least 20%, such as at least 40%, such as least 50%, such as at least 60%, like at least 70%, such as at least 80%, like at least 90%, such as at least 95%, like at least 98%, such as at least 99%.

55. The VLP according to any of claims according to any of claims 1-29 or 45 and / or a VLP produced by a method according to any of claims 40-44, and / or a composition as described in any of claims 30-32, for use as a medicament.

56. The VLP according to any of claims according to any of claims 1-29 or 45 and / or a VLP produced by a method according to any of claims 40-44, and / or a composition as described in any of claims 30-32 for use in the treatment or alleviation of a disorder treatable by prime editing.

57. The VLP according to any of claims 1-29 or 45 and / or a VLP produced by a method according to any of claims 40-44, and / or a composition as described in any of claims 30-32 for use in the treatment or alleviation of a disorder selected from the group consisting of monogenic diseases, such as, sickle cell disease, beta thalassemia, inborn errors of immunity (GATA2-deficiency (MonoMac), chronic- granulomatous disease, X-linked chronic granulomatous disease, hyper IgM syndrome, hyper IgE syndrome), cystic fibrosis, hemophilia, Wilson's disease, glycogen storage disease, hereditary angioedema, Duschenne muscular dystrophy, myotonic dystrophy type 1.

58. The VLP according to any of claims according to any of claims 1-29 or 45 and / or a VLP produced by a method according to any of claims 40-44, and / or a composition as described in any of claims 30-32 for use in the treatment or alleviation of a disorder selected from the group consisting of diseases of the eye, such as age-related macular degeneration, Leber's congenital amaurosis, and retinitis pigmentosa.

Images