WO2020225287A1 - Lentiviral nanoparticles - Google Patents

Abstract

The present invention relates to a lentivirus-based nanoparticle comprising a fusion protein comprising the following structure from N- to C-terminus: i. a Viral Protein R (Vpr), or a functional derivative thereof, ii. optionally a protease cleavage site and/or a linker, and iii. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof, a method for producing a lentivirus particle and its use for modifying genomic DNA sequences in target cells.

Description

LENTIVIRAL NANOPARTICLES

FIELD OF THE INVENTION

The invention generally relates to the field of viral nanoparticles, methods of their production and their use. The invention further relates to the packaging of cargo, such as foreign proteins and genes, into viral nanoparticles.

BACKGROUND OF THE INVENTION

Delivery of CRISPR/Cas RNA-guided endonucleases (RGEN) still poses a major challenge, limiting the scope and scalability of the RGEN systems. Lipofection, electroporation, nucleofection and virus-based techniques are widely used to deliver Cas9/sgRNA expression cassettes to recipient cells. As the DNA delivery methods suffer from undesired side effects such as limited cell-type specificity, integration into undesired chromosomal locations, immunogenicity, size-constrained packaging of expression cassettes (payload limit for AAV <4.7 kb) and increased off-targeting resulting from a sustained expression, the field has moved towards direct delivery of pre assembled Cas9 protein/sgRNA complexes (RNPs) to cells (1 , 2). A rapid turnover of RNPs limits the exposure of the genome to nucleases, thereby mitigating off-target effects. Furthermore, the transient occurrence of RNPs in cells is expected to elicit minimal innate and adaptive immune responses, especially when a synthetic, 5’triphospate-lacking sgRNA and a Cas9 ortholog derived from other than Streptococcus pyogenes bacterium, are used (3, 4). Although characteristics of the technology make RNP delivery advantageous compared to DNA transfer, it is restricted to cell types that do not suffer from reduced cell viability or phenotypic changes following chemical transfection or electroporation. Furthermore, the technology requires laborious optimization of transfection protocols for every cell type and lacks tissue- and cell- specificity. These shortcomings showed an urgent demand for a more versatile, safe, cell-selective and“easy-to-use” delivery system.

Lentiviral vectors (LVs), owing to their efficiency, low toxicity, simplicity of production, mild immunogenicity, relative safety, ease-of-use and possibility to customize cell tropism, have been commonly used in basic research and in gene therapy clinical trials (5, 6). LVs can also deliver foreign proteins of interest (POI) to mammalian cells (reviewed in, 7). Previous studies established a proof-of-concept that LVs can serve as platforms for the administration of“protein-based” designer nucleases to ablate host genes (8, 9). Modest effectiveness and, for RGENs, the need to simultaneously deliver both the programmable nuclease and sgRNA are the major challenges that need to be overcome to make of the system an applicable alternative to other RGEN delivery approaches.

Wu et al. incorporated heterologous proteins (staphylococcus nuclease (SN) or chloramphenicol transferase (CAT)) into virions as fusion proteins fused either to HIV Vpr or Vpx (21 , and US 2002/173643 A1 ).

Su et al. used fusion proteins comprising Vpr and a zinc finger protein (E2C) for site-specific incorporation of retroviral DNA in human cells (26).

WO 2019/050948 A1 discloses lentiviral particles for the delivery of a gene-editing fusion molecule, optionally together with a guide RNA. The gene-editing fusion molecule can be, for example, a fusion protein comprising a Cas protein and a Viral Protein R (Vpr).

Izmiryan et al. disclose two modes of meganuclease delivery, either as a protein or encoded by a vector. When the meganuclease is delivered as protein it is packaged into the virion as a fusion to Vpr, wherein cleavage sites for the HIV protease are introduced into the fusion protein to generate a Vpr-free meganuclease after processing inside the virion (8, and WO 201 1/007193 A1 ).

Choi et al. disclose the packaging of Cas9 protein fused to Gag protein into lentiviral particles. The fusion protein comprising Cas9 and Gag further comprises an HIV-1 protease cleavage site to allow the release of functional Cas9 protein during particle maturation (23).

Prior art viral nanoparticles, however, suffer from a major drawback, which is their poor efficiency for the packaging of cargo proteins, even when fused to viral proteins such as Vpr or Gag. Previous reports demonstrated a very poor encapsidation efficiency of directed endonucleases, such as zinc finger nucleases (ZFN) and TALEN nucleases (7) or meganucleases (8) resulting in poor genome editing efficiencies. Applications of lentiviral nanoparticles, such as targeted gene therapy using directed endonucleases, require efficient packaging of the POI to be able to deliver sufficient amounts of the protein or enzyme to the target. The POI has to resist the proteasomal cleavage within virions containing viral protease. Delivery of CRISPR/Cas RNA-guided endonucleases (RGEN), for example, still poses a major challenge, especially as both the programmable nuclease and guide (gRNA) have to be delivered simultaneously. Inefficient viral packaging thus limits the scope and scalability not only of the RGEN systems but also of many pharmaceutical and gene therapy-based applications where delivery of a POI or a transgene to a target cell is desired.

These shortcomings showed an urgent demand in the field for a more versatile, safe, cell-selective and“easy-to-use” delivery system.

SUMMARY OF THE INVENTION

It is the objective of the present invention to provide an improved system for the direct delivery of a protein of interest, such as an RNA guided DNA binding polypeptide (RgDBP), and in certain embodiments co-delivery of gRNA and a transgene. Specifically, it is the objective of the present invention to provide an efficient, target- specific and safe delivery system that is virtually unlimited regarding the choice of target cell and capable of delivering CRISPR enzyme to non-dividing cells. It is a further specific objective of the present invention to provide an efficient, target-specific and safe delivery system that is capable of delivering Clustered Regularly Interspaces Short Palindromic Repeats (CRISPR) enzyme, guide RNA (gRNA) and a transgene to a target cell.

The problem is solved by the subject matter of the present application.

As described herein, the presence of a rev response element (RRE) in the expression construct comprising the nucleic acid sequence encoding the protein of interest as well as in the expression construct(s) comprising viral genes, significantly increases the amount of POI encapsidated in the nanoparticle. Specifically, when RRE is comprised in the RNA transcripts comprising the POI as well as in the gag/pol transcripts, these transcripts and/or the translated proteins co-localize in the cytoplasm of the host cell. Co-localization then promotes interaction of POIs and viral proteins, leading to an increased amount of POI packaged into the nanoparticle. Furthermore, fusion of the POI to the viral protein Vpr ensures that the POI is co-encapsidated in the nanoparticle.

Fusion of an endonuclease to Viral Protein R (Vpr) allows efficient packaging of an RNA guided DNA binding polypeptide (RgDBP), specifically an endonuclease, into the viral particle, where it is released from Vpr by proteolytic cleavage. Modification of surface proteins of the lentivirus-based nanoparticles, such as for example fusion to antibodies, enables targeting of specific cells or cell populations, even non-dividing cells. Specifically provided herein is a significantly improved system for the co-delivery of endonuclease protein, specifically Cas9 protein, an RNA template encoding the guide RNA, and a transgene, to a target cell using a lentivirus-based nanoparticle.

According to the invention, there is provided a lentivirus-based nanoparticle comprising a fusion protein comprising the following structure from N- to C-terminus: i. a Viral Protein R (Vpr), or a functional derivative thereof,

ii. optionally a protease cleavage site and/or a linker, and

iii. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof.

Preferably, the fusion protein comprises the following structure from N- to C- terminus:

i. a Viral Protein R (Vpr), or a functional derivative thereof,

ii. a protease cleavage site,

iii. optionally a linker, and

iv. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof.

Specifically, fusion of an RNA guided DNA polypeptide to Vpr allows efficient packaging of the RNA guided DNA polypeptide into the nanoparticle described herein and does not interfere with the formation of said nanoparticle.

According to a specific embodiment of the invention, there is provided the nanoparticle described herein comprising the fusion protein described herein and one or more guide RNA (gRNA) templates comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or single guide RNA (sgRNA).

Specifically, there is provided a lentivirus-based nanoparticle comprising a) a fusion protein comprising the following structure from C- to N-terminus: i. a Viral Protein R (Vpr), or a functional derivative thereof,

ii. a protease cleavage site,

iii. optionally a linker, and

iv. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof; and

b) one or more guide RNA (gRNA) templates comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or single guide RNA (sgRNA).

According to a preferred embodiment, the RNA guided DNA binding polypeptide is selected from the group consisting of class 2 type II Cas proteins (e.g. Cas9), class 2 type V Cas proteins (e.g. Cpf1 , C2c1 , C2c3), class 2 type VI Cas proteins (e.g. Cas13a, Cas13b, Cas13c, Cas13d) class 1 type I Cas proteins (e.g. Cas3), class 1 type III Cas proteins (e.g.Casl O), class 1 type IV Cas proteins (e.g. Csf1 ), or a functionally active variant thereof. Preferably, the RNA guided DNA binding polypeptide is Cas9 or Cpf1 , or a functionally active variant thereof.

According to a further preferred embodiment, the fusion protein provided herein comprises SEQ ID NO:3 or SEQ ID NO:4, or a functionally active variant thereof. Specifically, said functionally active variant comprises at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO:3 or SEQ ID NO:4.

Specifically, the fusion protein further comprises a nuclear localization signal (NLS), preferably NLS SV40.

According to a specific embodiment, the nanoparticle provided herein comprises a) a fusion protein, specifically comprising SEQ ID NO:3 or SEQ ID NO:4, comprising the following structure from N- to C-terminus:

i. a Viral Protein R (Vpr),

ii. a protease cleavage site,

iii. a linker,

iv. a nuclear localization signal (NLS), and

v. a Cas 9 endonuclease; and

b) a sgRNA template.

Specifically, the protease cleavage site of the fusion protein provided herein is a human immunodeficiency virus (HIV) protease cleavage site.

Specifically, the nanoparticle described herein may comprise more than one gRNA template, specifically it comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 gRNA templates directed to the same or different target sequences. Specifically, the nanoparticle described herein may be used with gRNA templates of different sequences, targeting the same or different genomic sequences.

Specifically, the one or more gRNA templates are delivered to a target cell using the nanoparticle described herein comprising the fusion protein described herein, or using a different method, for example transfection or electroporation.

Specifically, the gRNA template is under control of an RNA polymerase III promoter, specifically a U6 or H1 promoter. According to a specific embodiment, the RNA guided DNA binding polypeptide is an endonuclease comprising an enzymatically inactive DNAse domain and is fused to a transcriptional regulator domain. Specifically, the transcriptional regulator domain can be a transcriptional activator or a transcriptional repressor.

According to a further specific embodiment, the RNA guided DNA binding polypeptide is an endonuclease fused to an enzyme comprising a deaminase domain, preferably selected from the group consisting of APOBEC1 , ADAR and TadA.

Specifically, Vpr or the functional derivative thereof as described herein is derived from a virus belonging to the genus of lentivirus, preferably which virus is selected from the group consisting of Human immunodeficiency virus (HIV), specifically HIV-1 or HIV- 2, Simian immunodeficiency virus (SIV), Bovine immunodeficiency virus (BIV), Caprine arthritis encephalitis virus, and Maedi-Visna virus.

According to a specific embodiment, the nanoparticle provided herein further comprises a transgene. Specifically, the transgene is a gene that is to be delivered to a target cell by the nanoparticle provided herein.

Specifically, the transgene is selected from the group consisting of a therapeutic gene, a reporter gene, a gene encoding an enzyme (e.g. catalase), a gene encoding a pro-drug enzyme, a gene encoding an apoptosis inducer, a gene encoding a suicide protein, a gene encoding an anti-immunosuppressive product, a gene encoding an epigenetic modulator, a gene encoding a T cell receptor (TCR), a gene encoding a chimeric antigen receptor (CAR), a gene encoding a protein that modifies the cell surface of transduced cells (e.g.CD52), a gene encoding a protein modifying the expression of the endogenous TCR, a gene encoding a switch receptor that converts pro-tumor into anti-tumor signals, a DNA segment encoding a pre-miRNA or shRNA, and a gene encoding a cytokine.

According to a specific embodiment, the nanoparticle described herein comprises a transgene which comprises a nucleic acid sequence encoding an RNA sequence capable of RNA interference, specifically pre-miRNA, miRNA, siRNA or shRNA. Specifically, the transgene is a DNA segment encoding a pre-miRNA or shRNA.

Advantageously, encapsidating the RNA guided DNA binding polynucleotide described herein in the nanoparticles described herein as protein instead of as nucleic acid, allows co-packaging of long nucleic acid sequences, such as e.g. therapeutic genes to be delivered to a target cell. Specifically, the nanoparticle provided herein comprises structural and enzymatic components, wherein the structural components preferably comprise a surface envelope protein, membrane, matrix capsid, nucleocapsid, and p6, and wherein the enzymatic components preferably comprise a reverse transcriptase, a protease and an integrase.

Further provided herein is the use of the nanoparticle described herein to modify a genomic DNA sequence in a target cell. According to a specific embodiment, said target cell can be a dividing and/or a non-dividing cell. In contrast to transfection or electroporation methods, virus-mediated delivery using nanoparticles is receptor- mediated and thus allows target-specific transfer to a variety of target cell populations. In some embodiments, the target cell is a mammalian cell with fast growth rates such as a mammal-derived cell line. In certain embodiments, the target cell is a mammalian somatic cell (i.e. other than reproductive cells), preferably in a living organism. Examples of somatic cells include various cell types of epithelial cells (e.g. keratinized epithelial cells, non-keratinized squamous epithelial cells, epithelial cells in the lungs, the gastrointestinal tract, the reproductive and urinary tract and cells of the exocrine and endocrine glands), muscle cells (skeletal, smooth and cardiac muscle cells), nerve cells (neurons and glial cells) and connective (e.g. fibroblasts, adipocytes, macrophages, leucocytes) tissue. In certain embodiments, the target cell is a progenitor cell (including undifferentiated stem cell and induced pluripotent stem cell) and a differentiated mature cell. Examples of progenitor cells include, but are not limited to, tissue stem cells, nerve stem cells, hematopoietic stem cells, mesenchymal stem cells and dental pulp cells. In some embodiments, the target cell is an animal-derived primordial germ cell and an animal-derived embryonic stem cell.

Specifically, the nanoparticle provided herein is used to modify a genomic DNA sequence in a target cell in vitro. Specifically, the target cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells, including human cells, and plant cells, preferably the target cells are human cells.

Specifically, the nanoparticle provided herein is used to modify a genomic DNA sequence in a target cell in vivo. Specifically, the target cell is selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells, and plant cells.

Specifically, the nanoparticle described herein is provided for use in the treatment of a disease. Specifically, the nanoparticle is used to modify a genomic DNA sequence or to deliver a therapeutic drug, pro-drug, nucleic acid, gene, protein and/or enzyme in a subject or patient. According to a specific embodiment, the nanoparticle provided herein is used to modify a genomic DNA sequence in a target cell ex vivo. An example of the target cell includes, but is not limited to, cells selected from the group of blood cells including hematopoietic stem cells and cells derived from myeloid and lymphoid progenitors, such as for example T-lymphocytes or macrophages.

In a further aspect, a method for treating a disease in a subject in need thereof is provided herein, wherein the method comprises the steps of:

a. providing a target cell;

b. contacting the target cell from step a. ex vivo with a therapeutically effective amount of any of the viral nanoparticles described herein or a pharmaceutical composition comprising such nanoparticles; and

c. administering the target cell from step b. to the subject.

Specifically, described herein is a method of producing a lentivirus-based nanoparticle, comprising the steps of introducing into a host cell:

i. a first expression construct encoding a transcript comprising

a. a nucleic acid sequence encoding a viral protein, specifically Viral Protein R (Vpr) or Gag,

b. optionally a nucleic acid sequence encoding a protease cleavage site and/or a linker between the sequences of a. and c.,

c. a nucleic acid sequence encoding a heterologous protein, and

d. at its 3’ end a stop codon followed by a Rev-response element (RRE), and ii. one or more second expression constructs comprising a gag gene followed by an RRE, a gag/pro/pol gene followed by an RRE, an env gene, and a rev gene; and iii. one or more further expression constructs comprising a gRNA template and/or a transgene, and an RRE; and

maintaining the host cell under conditions allowing the formation of the nanoparticle, followed by isolating the nanoparticle.

According to a preferred embodiment, the transgene and the gRNA template are comprised in the same expression construct, specifically they are comprised on the same expression vector.

Specifically, a vector comprising a gRNA template and/or a transgene comprises an RRE, which may be located downstream or upstream of the gRNA template and/or the transgene or in between the gRNA template and the transgene. Specifically, fusion of the protein of interest (POI), the heterologous protein, to Vpr or Gag allows packaging of the POI into the nanoparticle described herein and does not interfere with the formation of said nanoparticle.

Specifically, the nucleic acid sequence encoding a heterologous protein is a sequence encoding a polypeptide selected from the group consisting of RNA guided DNA binding polypeptides, preferably RNA guided endonucleases, Zinc-Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALEN), meganucleases and transposases.

Specifically, the one or more second and further expression constructs are selected from the group consisting of

i. an envelope vector comprising a nucleic acid sequence encoding an envelope protein, preferably VSV.G,

ii. a packaging vector comprising an RRE located 3’ to a nucleic acid sequence encoding Gag and GagProPol polyproteins, optionally including an integrase,

iii. a rev vector comprising a nucleic acid sequence encoding a Rev protein, iv. a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or guide RNA (gRNA) template, and an RRE,

v. a transgene vector comprising a nucleic acid sequence comprising a transgene and an RRE, and

vi. a vector comprising a gRNA template, a transgene and an RRE.

Specifically, the envelope vector, the packaging vector and/or the rev vector may be combined into one vector or two vectors.

Specifically, the RRE is derived from a virus belonging to the genus of lentivirus, preferably which virus is selected from the group consisting of Human immunodeficiency virus (HIV), specifically HIV-1 or HIV-2, Simian immunodeficiency virus (SIV), Bovine immunodeficiency virus (BIV), Feline immunodeficiency virus (FIV), Equine infectious anemia virus (EIAV), Caprine arthritis encephalitis virus (CAEV), and Maedi-Visna virus (MW).

Specifically, the RRE is derived from Human immunodeficiency virus. Even more specifically, the RRE comprises SEQ ID NO:5 or the RRE is a functionally active variant thereof comprising at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:5, or a functionally active variant thereof comprising 1 , 2, 3, 4, or 5 point mutations. According to a further specific embodiment, the RRE comprises SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35, or the RRE is a functionally active variant thereof comprising at least 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35, or a functionally active variant thereof comprising 1 , 2, 3, 4, or 5 point mutations.

According to a preferred embodiment, the method of producing the lentivirus- based nanoparticles described herein, comprises the following, preferably sequential, steps of

i. introducing into a host cell the following DNA vectors:

a. an endonuclease vector comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a functional derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably a Cas9 endonuclease,

b. a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or guide RNA (gRNA), and an RRE, and

c. one or more vectors comprising a gag gene, a pol gene, an env gene, a rev gene and, optionally, an RRE,

ii. maintaining the host cell under conditions to allow formation of the nanoparticles, and

iii. isolating the nanoparticles.

Specifically, the gag, pol, env and rev genes are derived from a virus, preferably from a lentivirus. Specifically, the gag, pol, env and rev genes are artificial nucleic acid sequences, specifically wherein said sequence is derived from a virus, such as a lentivirus. Specifically, the gag, pol, env and/or rev genes are lentiviral genes.

According to a specific embodiment, the method described herein comprises the steps of

i. introducing into a host cell the following DNA expression constructs:

a. an endonuclease vector comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a functional derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably a Cas9 endonuclease, b. a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or guide RNA (gRNA), an RRE, and optionally a transgene, and

c. an envelope vector comprising a nucleic acid sequence encoding an envelope protein, preferably VSV.G,

d. a packaging vector comprising an RRE and a nucleic acid sequence encoding Gag and GagPol polyproteins, optionally including an integrase, and

e. a rev vector comprising a nucleic acid sequence encoding a Rev protein, ii. maintaining the host cell under conditions to allow formation of the nanoparticles, and

iii. isolating the nanoparticles.

Specifically in the method provided herein, one or more of the DNA vectors comprise an RNA Polymerase II promoter, preferably one or more of Cytomegalovirus (CMV) promoter, CAG promoter or Rous-Sarcoma-Virus (RSV) promoter. Specifically, any RNA Polymerase II promoter may be used, such as for example elongation factor 1a promoter, elongation factor 1a short (EFS) promoter, or simian virus 40 (SV40) promoter, spleen focus forming virus (SFFV) promoter, ubiquitin promoter, or phospho- glycerate kinase (PGK) promoter.

Specifically, the gRNA template vector comprises at least one RNA Polymerase III promoter, preferably a U6 or H1 promoter. Specifically, one or more of the vectors comprise a polyadenylation signal.

According to a specific embodiment of the nanoparticle provided herein, and of the method of its production as provided herein, the integrase is a catalytically inactive integrase. Preferably, if the nanoparticle described herein comprises a transgene for delivery to a target cell, the nanoparticle comprises a catalytically active integrase.

Further provided herein is a kit comprising the following plasmids:

i. an endonuclease plasmid comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably Cas9 endonuclease,

ii. an RNA template plasmid comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA) and an RRE,

iii. one or more plasmids comprising a gag gene, a pol gene, an env gene, a rev gene and, optionally, an RRE. Specifically, the gag, pol, env and rev genes of the plasmids comprised in the kit provided herein are derived from a virus, preferably from a lentivirus. Specifically, the gag, pol, env and rev genes are artificial nucleic acid sequences, specifically wherein said sequence is derived from a virus, such as a lentivirus.

According to a specific embodiment, the kit described herein comprises a plasmid comprising a transgene as described herein, and an RRE. Preferably, the transgene is present on the gRNA template plasmid.

Further provided herein are polynucleotides encoding the nanoparticle described herein, or parts thereof, and cell lines comprising said polynucleotides. Specifically, said cell lines comprising said polynucleotides are host cell lines comprising one or more vectors comprising said polynucleotides for transcription into the components of the nanoparticles described herein. Specifically, after formation of the lentivirus-based nanoparticles described herein in said cell lines, mature nanoparticles are harvested, preferably from the supernatant. Further provided herein are thus cell lines, comprising the lentivirus-based nanoparticles described herein.

Specifically provided herein is an expression construct encoding a transcript comprising

i. a nucleic acid sequence encoding Viral Protein R (Vpr),

ii. optionally a nucleic acid sequence encoding a protease cleavage site and/or a linker between the sequences of i. and iii.,

iii. a nucleic acid sequence encoding a heterologous protein, specifically an RNA guided DNA binding polypeptide, and

iv. at its 3’ end a stop codon followed by a Rev-response element (RRE),

wherein the RRE directs nuclear export of the transcript and its localization to a cytoplasmic microdomain.

According to a specific embodiment, the expression construct comprises the following sequence from 5’ to 3’ end:

i. a nucleic acid sequence encoding Vpr,

ii. a nucleic acid sequence encoding a protease cleavage site,

iii. a nucleic acid sequence encoding a heterologous protein, preferably an RNA guided DNA binding polypeptide, and

iv. a stop codon followed by an RRE.

Specifically, the transcript produced from the first expression construct, encoding the heterologous protein and an RRE as described herein, is directed by said RRE into the cytoplasm of the host cell. Importantly, the transcript(s) produced from the second and further expression constructs described herein, specifically those encoding proteins, enzymes and nucleic acids required for the production of the nanoparticle described herein, are co-localized with the transcript originating from the first expression construct in the cytoplasm of the host cell. Further provided herein is a pharmaceutical composition, comprising the lentivirus-based nanoparticle described herein. Specifically provided herein is a method of producing said pharmaceutical composition, comprising formulating the lentivirus-based nanoparticle described herein with a pharmaceutically acceptable carrier.

FIGURES

Figure 1. Delivery of bi-component lentivector nanoparticles carrying Cas9 nuclease protein and a template for U6-sgRNA expression cassette to human HEK293- EGFP cells. (A) Design of constructs used to generate the lentivector articles. (B) A schematic representation of lentivector-mediated delivery of Cas9 protein and viral RNA containing U6-sgRNA.

Figure 2. (A) Integration site as determined by Sanger sequencing of the LM- PCR product (linker sequence is shown in bold, human sequence in italics and vector sequence is underlined). (B) EGFP gene disruption in HEK293-EGFP cells after transduction with the bi-component lentivectors (black entries) or a control LentiCRISPRv2(sgGFP) (white entry).

Figure 3. (A) Indels in EGFP gene resulting from transductions with the two- component lentivector, the same vector lacking Vpr.Prot.Cas9 or the control pLentiCRISPRv2(sgGFP). (B) Mutant sequences at the EGFP locus and their frequencies. The 20-nt target sequence is shown with a grey background. The PAM sequence is shown in bold.

Figure 4. Determination of EGFP gene editing efficiency by Inference of CRISPR Edits (ICE from SYNTHEGO). (A) Summary of editing results. (B) Sanger sequencing chromatograms of the edited (upper panel) and control (lower panel) samples. (C) The left panel shows the level of disagreement between the control and edited samples around the cut site. Distribution of indel sizes and their frequencies is depicted in the right panel.

Figure 5. Transduction with two-component lentivector has minimal effect on cell viability. (A) Cell viability was measured by the Cell Proliferation (XTT) assay (Roche) three days post transduction with VECTRv2(sgGFP). Mean viabilities of two biological replicates are shown. Error bars, mean ± SD. (B) Quantification of formazan dye formed from XTT tetrazolium salt added to different amounts of cells seeded in 12 well plates. Mean absorbance values ± SD from two replicates are shown. (C) Titration of the pVpr.Prot.Cas9 plasmid for optimal gene disruption activity.

Figure 6. Comparison of EGFP disruption after transduction with lentiviral particles containing integration deficient (D64V) or proficient (WT) integrase.

Figure 7. (A) Time-course analysis of EGFP disruption mediated by the bi component VECTR(sgGFP) or the gene-delivering LentiCRISPRv2(sgGFP). (B) Flow cytometry to determine the percentage of EGFP-expressing cells at various time points after transduction that was then used for the calculation of disruption activity in (A). (C) EGFP expression in HEK293EGFP cells transduced with EGFP- or EMX1 -targeting bi component VECTRv2 was measured over a period of 35 days post transduction.

Figure 8. Double mismatch tolerance of the bi-component VECTRv2(sgGFP) vs. LentiCRISPRv2(sgGFP) harboring variant mismatched sgRNAs. (A) The targeted EGFP sequence. (B) EGFP gene disruption in HEK293-EGFP cells after transduction with the bi-component lentivectors (black entries) or a control LentiCRISPRv2(sgGFP) (white entry). The gRNAs contained mismatched dinucleotides. The gRNA with sequence matching the target locus was used as a control.

Figure 9. (A) Detection of indels by T7E1 assay performed on endogenous EMX1 , FANCF, HEKsl , HEKs3 loci in HEK293-EGFP cells transduced with VECTRv2(sgRNA) or LentiCRISPRv2(sgRNA). (B) VECTRv2(sgRNA)-mediated mutations on FANCF, HEKsl and HEKs3 loci in lines of human T (Jurkat, SupT1 ) and B (IM9) lymphocytes, and monocytic cell line (THP-1 ) as measured by T7E1 assay. Mutation rates obtained from parallel transductions of HEK293-EGFP cells are also shown. (C) NHEJ rates (measured by T7E1 assay) in primary CD4+ T cells transduced with VECTRv2(sgRNA). (D-E) NHEJ frequencies as quantified by CRISPR Genome Analyzer using next-generation sequencing data of amplicons from (C) as an input. (D) SupT1 T cell line; (E) primary CD4+ T cells.

Figure 10. EGFP disruption activity of lentivectors carrying either Gag.Cas9 or Vpr.Pro.Cas9 fusion protein.

Figure 11. Nucleotide and amino acid sequences referred to herein, in particular Vpr.Prot.Cas9. Figure 12. Design of constructs used to generate the viral nanoparticles of Example 9.

Figure 13. Immunoblot analysis of lentivector nanoparticles and lysates of cells producing lentivector nanoparticles.

Figure 14. Schematic representation of the packaging of viral nanoparticles using the methods and constructs described herein.

Figure 15. Schematic representation of viral nanoparticles comprising Cas9 protein and a transgene.

Figure 16. Design of constructs used to generate the viral nanoparticles of Example 10.

Figure 17. Simultaneous knock-out of an endogenous gene (eg/p) and delivery of a transgene ( DsRed ). HEK293GFP cells carrying a single copy of egfp gene were transduced with three-component lentivector nanoparticles VECTRv2-Cas(sgGFP)- DsRed containing the Cas9 protein, a template for sgRNA targeting the egfp gene (sgGFP), and the DsRed transgene under the control of EF1 a promoter. As a control the cells were transduced with control lentiviral nanoparticles (VECTRv2-Cas(sgEMX)- puro). The EGFP and DsRed expression was monitored by flow cytometry and UV microscopy three days post transduction. The expression of EGFP was disrupted in >80% of HEK293EGFP cells transduced with VECTRv2-Cas(sgGFP)-DsRed. Simultaneously, 99% of the transduced cells expressed DsRed (left figures). In contrast, transduction with the control lentivector did not result in a reduction of the EGFP expression (98% of cells produced EGFP) (right figures).

Figure 18. Cytotoxicity of T cells (SupT1 cell line) transduced with lentivector nanoparticles VECTRv2-Cas(sgGFP)-CAR[CD19] against SupT1 cells that were engineered to express the CD19 and firefly luciferase (SupT1 CD19-luc). The lentivector was used for parallel transductions of HEK293EGFP and SupT1 cells. As shown in Figure 18 (A), the transduction of the HEK293EGFP cells resulted in a loss of the EGFP expression in ~98% of cells. (B) The SupT1 cells transduced with three-component lentivector nanoparticles containing the Cas9 protein, a template for the expression of sgRNA targeting egfp (sgGFP), and the transgene encoding a chimeric antigen receptor specific for CD19 (CAR[CD19]) expressed the CAR[CD19] on the cell surface. This is evidenced by the cytotoxicity of the cells against the cells expressing CD19 antigen (SupT1 CD19-luc). When the transduced cells (effector) were mixed with SupT1 CD19- luc (target) at ratios ranging from 1 :1 to 4:1 , a loss of the firefly luciferase expression (cell killing) was observed. Co-cultivation of the untransduced cells with the target cells did not lead to a reduction of the luciferase expression. SupT1 CD19-luc cells treated with 1 % Triton X-100 (complete lysis) or media alone (spontaneous lysis) served as experimental controls. Following 24-hour co-culture, cell extracts were made using the cell culture lysis reagent and substrate was added according to instructions for the Luciferase Assay System (Promega). The percentage of killing was calculated as follows: (experimental - spontaneous lysis) x 100 / (complete - spontaneous) lysis.

Figure 19. Delivery of the first generation of the two-component lentivector nanoparticles carrying the Cas9 nuclease protein and a template for the U6-sgRNA expression cassette (VECTR-Cas(sgGFP)) to human HEK293-EGFP cells. (A) Design of the constructs to generate the lentivector particles. Cas9 was fused to the C-terminus of Vpr containing an authentic HIV-1 protease cleavage site (CTLNF/PISPI; Vpr.Prot.Cas9). The U6-sgRNA expression cassette was incorporated into a lentiviral expression vector (Lenti(sgRNA)). The packaging construct (psPAX2) encodes the structural and enzymatic components of virions. The VSV.G envelope protein was used to pseudotype and stabilize viral particles (pHCMV-G). Efficient nuclear export and colocalization of mRNA for translation were supported by adding the Rev-responsive element (RRE) to the constructs and by overexpressing Rev during virion production (pRSV-Rev). Gag-Pol subunits: matrix (MA), capsid (CA), nucleocapsid (NC), p6, reverse transcriptase (RT), and integrase (IN). Packaging signal (y); promoters (CMV, CAG, RSV, U6, and EFS), polyadenylation signal (pA), post-transcriptional regulatory element (WPRE). (B) The EGFP gene disruption in HEK293-EGFP cells after transduction with the first generation of the two-component lentivector (VECTR- Cas(sgGFP); left column) or a control LentiCRISPRv2(sgGFP) (right column).

Figure 20. Delivery of the second generation of the two-component lentivector nanoparticles carrying the Cas9 nuclease protein and a template for the U6-sgRNA expression cassette (VECTRv2-Cas(sgGFP)) to human HEK293-EGFP cells. The same experiment as in Figure 19, except the employment of a modified Vpr.Prot.Cas9 expression cassette containing the RRE.

DETAILED DESCRIPTION

Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin^'s Genes XI", Jones & Bartlett Learning, (2017), and Murphy & Weaver, "Janeway ^'s Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.

The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wild- type) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, fusion constructs, expression constructs, transformed host cells and modified proteins, are“man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.

The terms“comprise”,“contain”,“have” and“include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements.“Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus“comprising” is broader and contains the“consisting” definition.

The term“about” as used herein refers to the same value or a value differing by +/-5 % of the given value.

As used herein and in the claims, the singular form, for example“a”,“an” and “the” includes the plural, unless the context clearly dictates otherwise.

The term “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats) or“CRISPR/Cas” as used herein with respect to a CRISPR enzyme such as the RNA guided DNA binding polypeptide described herein, shall refer to an enzyme which can be used in a CRISPR/Cas system (also referred to as“CRISPR system”). Generally, CRISPR/Cas systems require an RNA guided DNA binding polypeptide such as a Cas enzyme and a CRISPR RNA (crRNA), or guide RNA.

According to specific embodiments, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Streptococcus thermophilus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Specifically, such elements are an RNA guided DNA binding polypeptide as described herein and a guide RNA as described herein.

The term “RNA guided DNA binding polypeptide” or “RgDBP” refers to a polypeptide or protein comprising DNA binding activity and specifically also endonuclease activity. Specifically, an RgDBP described herein comprising endonuclease activity comprises one or more active DNase domain(s).

In some embodiments described herein, the RgDBP is an endonuclease variant comprising an inactive DNAse domain, substantially lacking all DNA cleavage activity e.g., the DNA cleavage activity of the mutated polypeptide is less than about 25%, 10%, 5%, 1 %, 0.1 %, 0.01 %, or lower with respect to its non-mutated form. For example, the RgDBP has no detectable DNAse activity, or less than 1 % of the nuclease activity of the non-mutated or wild type RNA-guided deoxy-ribonuclease. Specifically, the DNAse domain may be inactivated by introduction of one or more mutations, preferably point mutations, into the sequence of the DNAse domain.

In specific embodiments, a RgDBP which is lacking nuclease activity is further modified to enable programmable transcriptional regulation, specifically by fusion to a transcriptional regulator domain. For example, fusing an activation domain (e.g. VP64) to the polypeptide renders the polypeptide transcriptionally active. In further examples, fusing a repressor domain (e.g. KRAB) to the RNA guided DNA binding polypeptide renders it transcriptionally repressive.

According to a specific embodiment, the RgDBP is a“CRISPR enzyme”, (or Cas enzyme). Specifically, said Cas enzyme is directed (guided) to at least one target nucleotide sequence by a gRNA as described herein.

The Cas locus has more than 50 gene families and 395 profiles for 93 Cas proteins have been identified. Classification includes signature gene profiles plus signatures of locus architecture. CRISPR/Cas systems fall into two classes. Class 1 , divided into types I, III and IV, includes multi-subunit crRNA-effector complexes and class 2, divided into types II, V and VI, includes single-subunit crRNA-effector complexes.

According to a specific embodiment, the RNA guided DNA binding polypeptide described herein is of a class II Type II CRISPR/Cas system, for example Cas9. Typically, endonucleases of a class II Type II CRISPR/Cas system comprise the nuclease domains HNH and RuvC, which together can produce double strand breaks (DSBs) or individually can produce single strand breaks. According to a further specific embodiment, the RNA guided DNA binding polypeptide described herein is of a class II Type V CRISPR/Cas system, for example Cpf1. Endonucleases of a class II Type V CRISPR/Cas system lack the HNH domain, required by class II Type II Cas enzymes such as Cas9, and harbor a single DNAse domain RuvC that catalyzes cleavage of a double stranded DNA substrate. Specifically, Cpf1 is generally smaller than most of Cas9 orthologs and can therefore easily be packaged into viral nanoparticles. Cpf1 recognizes a protospacer adjacent motif (PAM) sequence with a high frequency in the genome, which is beneficial for clinical purposes. Since its PAM sequence occurs at a higher frequency, it will be found in proximity to many therapeutic genes.

A variety of native (wild-type, unmodified) CRISPR enzymes are well-known, such as for example class 2 type II Cas proteins (e.g. Cas9), class 2 type V Cas proteins (e.g. Cpf1 , C2c1 , C2c3), class 2 type VI Cas proteins (e.g. Cas13a, Cas13b, Cas13c, Cas13d) class 1 type I Cas proteins (e.g. Cas3), class 1 type III Cas proteins (e.g.Casl O) or class 1 type IV Cas proteins (e.g. Csf1 ).

According to further specific examples, the RgDBP described herein is fused to a transcriptional regulator domain which is a base editor, e.g., a deaminase that modifies cytosine DNA bases, e.g., a cytidine deaminase from the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, including APOBEC1 , APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D/E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, an adenosine deaminase 1 (ADAR1 ), ADAR2 or naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

Specifically, the components sufficient to generate a nucleic acid-targeting complex, such as the RgDBP system, specifically the CRISPR system described herein, including the CRISPR enzyme, and one or more gRNA templates, are delivered to a target cell having the corresponding target (genomic) nucleic acid sequence using the lentiviral nanoparticles provided herein.

According to further specific embodiments, the “heterologous protein”, also referred to as“protein of interest” or“POI”, comprised in the fusion protein described herein can be an RNA guided DNA binding polypeptide as described above, or a zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease and/or transposase. Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut target sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, Fokl. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences.

Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that assist targeted editing of the genome by creating double strand breaks (DSBs) in DNA at targeted locations. ZFNs comprise two functional domains: i) a DNA-binding domain comprising a chain of two finger modules (each recognizing a unique hexamer (6 bp) sequence of DNA - two-finger modules are stitched together to form a Zinc Finger Protein, each with specificity of at least 24 bp) and ii) a DNA-cleaving domain comprising a nuclease domain of Fokl. When the DNA binding and -cleaving domains are fused together, a highly-specific pair of "genomic scissors" are created.

In certain embodiments, each monomer of the ZFN comprises 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain binds to a 3 bp subsite. In other embodiments, the ZFN is a chimeric protein comprising a zinc finger-based DNA binding domain operably linked to an independent nuclease. In certain embodiments, the independent endonuclease is a Fokl endonuclease. In certain embodiments, the gene-editing molecule comprises a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably linked to a Fokl nuclease, wherein the first and the second ZFN recognize two contiguous target DNA sequences in each strand of the target DNA sequence separated by about 6 bp to about 40 bp cleavage site or about a 5 bp to about 6 bp cleavage site, and wherein the Fokl nucleases dimerize and make a double strand break.

ZFNs and TALENs introduce DSBs in a target genomic sequence and activate non-homologous end-joining (NHEJ)-mediated DNA repair, which generates a mutant allele comprising an insertion or a deletion of a nucleic acid sequence at the genomic locus of interest and thereby causes disruption of the genomic locus of interest in a cell. DSBs also stimulate homology-directed repair (HDR) by homologous recombination if a repair template is provided. HDR can result in a perfect repair that restores the original sequence at the broken site, or it can be used to direct a designed modification, such as a deletion, insertion, or replacement of the sequence at the site of the double strand break.

In still another embodiment, the heterologous protein comprised in the fusion protein is a meganuclease. Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG (SEQ ID NO:36), GIY- YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. Meganuclease domains, structure and function are well known in the art.

In a certain embodiment, the heterologous protein comprised in the fusion polypeptide can be a transposase. Transposases bind to the transposon sequences, which may contain a transgene expression cassette, and catalyze their insertion to the host genome. Examples of transposase proteins include, but are not limited to, Sleeping Beauty and piggyback DNA transposases and their hyperactive variants such as SBI OOx and hyPBase.

As used herein, the term "guide RNA" or "gRNA" comprises any polynucleotide sequence having sufficient complementarity with a target nucleotide sequence to hybridize with the target nucleotide sequence. Specifically, the guide RNA has sufficient sequence complementarity to effect sequence specific binding of a nucleic acid-targeting complex, which comprises at least an RNA guided DNA binding polypeptide as described herein and a gRNA, to the target nucleotide sequence. Upon sequence- specific binding of the nucleic acid-targeting complex to the target nucleotide sequence, the CRISPR complex is formed. The gRNA referred to herein therefore typically comprises crRNA (CRISPR RNA) and tracrRNA (trans-acting CRISPR RNA) and/or sgRNA (single-guide RNA).

In the natural S. pyogenes system, the guide RNA is actually a complex of two RNA polynucleotides, a first CRISPR RNA (crRNA) containing about 20 nucleotides that determine the specificity of the Cas9 enzyme as well as the trans-activating CRISPR RNA (tracrRNA) which hybridizes to the crRNA to form an RNA complex that interacts with Cas9. The terms crRNA and tracrRNA are also known as tracr-mate RNA and tracr RNA. Instead of the natural two part guide RNA complex, engineered single polynucleotide guide RNAs (sgRNA) are successfully used by CRISPR-based methods.

According to a specific embodiment, a guide RNA as described herein comprises a guide sequence and a direct repeat sequence, wherein said guide sequence is capable of hybridizing to a target nucleotide sequence.

Guide RNAs described herein may have a length of 10 to 500 nucleotides.

Specifically, a guide sequence within a gRNA is complementary to a target sequence to which the gRNA is directed. Specifically, the guide sequence is about or more than about 5, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably, the guide sequence is 10 to 30 nucleotides long, even more preferably 15 to 25 nucleotides long and most preferably 20 or 21 nucleotides long. In general, a guide sequence of a gRNA is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and to direct sequence-specific binding of a CRISPR complex to the target sequence.

The term“gRNA template” as used herein refers to a nucleotide sequence that is a template of a gRNA described herein. According to a specific embodiment, the gRNA template is integrated in the lentivirus-based nanoparticle described herein. In the nanoparticle, the gRNA is part of the viral RNA, which, upon integration of the nanoparticle into a target cell, is reverse transcribed into DNA. Following reverse transcription, the gRNA is produced in the target cell, specifically in the nucleus. Specifically, the DNA encoding the gRNA, which has been generated by reverse transcription of the gRNA template, can be stably integrated into the target cell’s genome.

Specifically, using the system for co-delivery, as described herein, the CRISPR enzyme and the gRNA template, as well as the transgene, remain embedded in a lentiviral capsid even after receptor-facilitated cell entry of the nanoparticle. Following nuclear entry, the lentiviral capsid releases its cargo. This has the advantage that the CRISPR enzyme and the gRNA template, and also the transgene, are protected from degradation and co-delivered into the nucleus of the target cell where the CRISPR complex is formed. As used herein, the term “nanoparticle” refers to an engineered particle, comprising viral components and encapsidated heterologous protein, such as the fusion protein described herein. According to specific embodiments, the nanoparticle described herein comprises the fusion protein described herein and the transgene described herein. The nanoparticle is specifically a membrane vesicle, which is a sub- microscopical vesicle comprising a lipid membrane. Specifically, the nanoparticle is produced by cells or synthetically produced. When produced by a cell, the nanoparticle is released by the cell into the extracellular space by biological processes, and the fusion protein described herein is engineered prior to the cell release. The nanoparticle may be surface decorated e.g., with carbohydrate structures and/or by fusion with amino acids or amino acid sequences, such as antibodies, and/or by coupling with chemical compounds, such as drugs, labels or tags. Specifically, the nanoparticle provided herein has a size of about 80-150 nm in diameter, specifically it has a size of about 80, 90, 100, 1 10, 120, 130, 140 or 150 nm in diameter.

According to a preferred embodiment, the nanoparticle provided herein is lentivirus-based, meaning most or all of its components are derived from a lentivirus. The lentivirus belongs to the retroviridae family of viruses. Following the transduction of a host cell, retrovirus RNA is reverse-transcribed into viral cDNA. Lentiviruses can integrate a significant amount of viral cDNA into the DNA of the host cell and can become endogenous, by integrating their genome into the host cell’s germline genome. After integration, the host cell will transcribe the viral genes along with its own genes, thereby producing stable transgene expression. Specifically, lentiviruses are unique members of the retroviridae family as most retroviruses cannot productively infect non-dividing cells whereas lentiviruses can infect cells regardless of their proliferation status, making them particularly attractive for human gene therapy. Hepatocytes, neurons, hematopoietic stem cells, monocytes, and macrophages are some examples of potential target cells for lentiviral vector based gene therapy.

Specifically, the nanoparticles described herein comprise structural and enzymatic components that are typically encoded by the viral, specifically lentiviral, gag, pol and env genes.

Specifically, the one or more second expression constructs described herein comprise (a) GAG, (b) POL, and/or (d) REV retroviral (e.g., lentiviral) elements, each of which may be considered involved with the assembly of the viral nanoparticle. In some embodiments, the one or more second expression constructs comprise an envelope plasmid. In certain embodiments, an envelope plasmid comprises (a) VSV -G, Ebola virus envelope, MLV envelope, Measles virus envelope, GALV envelope, RD1 14 envelope, LCMV envelope, Rabies virus envelope and/or (b) PolyA.

In certain embodiments, the envelope of the retroviral particle can be pseudotyped. Pseudotyping is to alter the tropism of the nanoparticle or for generating increased or decreased stability of a viral nanoparticle. As such, foreign viral envelope proteins (heterologous envelope proteins) are introduced into the nanoparticle. They are typically glycoproteins derived from portions of the membrane of the virus infected host cells or glycoproteins encoded by the virus genome. The structural envelope proteins (e.g., Env) can determine the range of target cells that can ultimately be infected and transformed by recombinant retroviruses. In the case of lentiviruses, such as HIV -1 , HIV -2, SIV, FIV and EIV, the Env proteins include gp41 and gp120. When producing recombinant retroviruses (e.g., recombinant lentiviruses), a wild type retroviral (e.g., lentiviral)) env, gene can be used, or can be substituted with any other viral env, gene from another lentivirus or other virus (such as vesicular stomatitis virus GP (VSV -G)) or an artificial chimeric envelope protein as described for example in Anliker, B., et al. (2010) Nat Methods 7(1 1 ):929-935.

The gag gene specifically encodes the Gag precursor polypeptide which is matured, specifically by cleavage by a viral protease, into structural components of mature virions. Specifically, Gag is a polyprotein that is processed into matrix and other core proteins that determine retroviral core. The Gag proteins form the viral core structure, RNA genome binding proteins, and are the major proteins comprising the nucleoprotein core particle. Specifically, the structural components comprise matrix (MA) proteins, capsid (CA) proteins, nucleocapsid (NC) proteins and p6. Typically, MA proteins aid in virion assembly and infection of non-dividing cells, CA proteins form the hydrophobic core of virion and nucleocapsid proteins protect the viral genome by coating and associating tightly with the viral RNA.

The pol gene specifically encodes for the viral protease (PRO), reverse transcriptase (RT) and integrase (IN), enzymes aiding in viral replication. Specifically, the Pol polyprotein comprises reverse transcriptase, RNase H and integrase functions.

Specifically, the env gene encodes surface proteins, specifically the viral surface glycoprotein gp160, which is cleaved into the surface protein gp120 (SU) and the transmembrane protein gp41 (TM) during the process of viral maturation. Specifically, these surface proteins are essential for virus entry into the host cell as they enable binding to specific cellular receptors and fusion with cellular membranes. Specifically, Env is the envelope protein, which resides in the lipid layer of the viral nanoparticle.

Specifically, the rev gene comprises a nucleic acid sequence encoding the Rev protein and a nucleic acid sequence encoding a nuclear localization signal, which allows the Rev protein to be localized to the nucleus. Specifically, nucleic acid sequences, specifically mRNA, comprising a Rev-Response Element are exported out of the nucleus by the Rev protein. Specifically, nucleic acid sequences comprising an RRE are co localized in the cytoplasm.

Specifically, the gag, pol, env and rev genes described herein are derived from a virus, preferably from a lentivirus. Specifically, the gag, pol, env and rev genes are artificial nucleic acid sequences, specifically wherein said sequence is derived from a virus, such as a lentivirus.

Specifically, the nanoparticle described herein is derived from an attenuated lentivirus. The term“attenuated” is used herein to describe a virulent strain of lentivirus that has been modified so that it is no longer capable of causing disease, i.e. , the modified strain is avirulent. The lentiviral strain is“live” in that it is able to grow and reproduce in the host cell and capable of assembling the lentiviral-based nanoparticles provided herein.

The terms“Viral Protein R” or“Vpr” as used herein, refer to a lentiviral protein, belonging to the so-called class of accessory proteins. Specifically in HIV, Vpr plays an important role in regulating nuclear import of the HIV-1 pre-integration complex, and is required for virus replication in non-dividing cells. Importantly, Vpr is not a structural protein and thus not required for correct formation of lentiviral particles. Preferably, the Vpr described herein is a recombinant protein, fused to the RNA guided DNA binding polypeptide described herein via a protease cleavage site.

Specifically, Vpr is assembled into virions by interaction with the p6 domain of Gag precursor, thereby mediating encapsidation of its fusion partner into the nanoparticle provided herein.

According to a preferred embodiment, the Vpr of the fusion protein described herein comprises SEQ ID NO:7 or a functional derivative thereof, preferably comprising at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:7. As used herein the term“fusion protein” refers to an RNA guided DNA binding polypeptide as described herein, which is linked to a Viral Protein R as described herein and optionally comprises a protease cleavage site as described herein between the RNA guided DNA binding polypeptide and the Vpr. Specifically, the RNA guided DNA binding polypeptide is covalently linked to the protease cleavage site which is covalently linked to the Vpr. Preferably, the fusion protein comprises at least one linker. Specifically, a linker may include any amino acid sequence that does not interfere with the function of elements being linked. The linkers may be used to engineer appropriate amounts of flexibility. Preferably, the linkers are short, e.g., 2-20 nucleotides or amino acids, and are typically flexible. Amino acid linkers commonly used consist of a number of glycine, serine, and optionally alanine, in any order. Such linkers usually have a length of at least any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acids or more, as required. In some embodiments, the linker comprises one or more units, repeats or copies of a motif.

Fusion of an RNA guided DNA binding polypeptide described herein to Vpr allows efficient packaging of the CRISPR enzyme into the nanoparticles described herein. This system allows direct delivery of Cas9 protein, optionally together with gRNA templates, to specific target cells to mediate target-specific and efficient modification of gene expression with few to no side-effects. Advantageously, fusing the RgDBP, specifically the CRISPR enzyme, to Vpr instead of a structural component comprised in the lentiviral Gag polypeptide avoids structural disturbances in the formation of the nanoparticles, which could lead for example to reduced transducibility.

The term“protease cleavage site” refers to a sequence of amino acids within the fusion protein described herein, which is specifically recognized and cut by a protease. The protease may be a viral protease or any other type of natural or artificial, e.g. recombinant, protease. Preferably, the protease cleavage site is a sequence that is specifically recognized by a retroviral protease, preferably a lentiviral protease. According to a specific example, the protease cleavage site is any of the amino acid sequences recognized and cut by the HIV-1 protease (PR), a retroviral aspartyl protease. A specific example of the protease cleavage site is SEQ ID NO:8.

Specifically, integration of a protease cleavage site that is recognized and cut by a lentiviral protease, such as PR, causes separation of the RgDBP, specifically the CRISPR enzyme from the Vpr in the mature lentivirus-based nanoparticle described herein and thereby avoiding any potential sterical hindrance of the RgDBP, specifically the CRISPR enzyme by Vpr. Accord ing to a specific embodiment, the fusion protein described herein does not comprise a protease cleavage site and the complete fusion protein is introduced into the target cell.

According to a specific embodiment, the lentivirus-based nanoparticle provided herein is produced according to a method comprising the steps of

i. introducing into a host cell the following DNA vectors:

a. an endonuclease vector comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a functional derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably a Cas9 endonuclease;

b. optionally, a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA) and an RRE, and optionally a transgene; and

c. one or more vectors comprising a lentiviral gag gene, a pol gene, an env gene, a rev gene and, optionally, an RRE,

ii. maintaining the host cell under conditions to allow formation of the nanoparticles, and

iii. isolating the nanoparticles.

According to the method provided herein, vectors comprising polynucleotides encoding components of the nanoparticles provided herein preferably comprise a Rev- response element. The term“Rev-response element” or“RRE”, as used herein, refers to a nucleic acid sequence promoting export of a nucleic acid transcript from the nucleus into the cytoplasm. Specifically, RRE aids nuclear export of mRNA comprising such RRE into the cytoplasm, in the presence of Rev. RRE in combination with the accessory protein Rev, enhances cytoplasmic mRNA levels of the nanoparticle components and directs the mRNA for translation at the same intracytoplasmic microdomains, enhancing the efficiency of nanoparticle assembly and integration of the fusion protein into said nanoparticle. Specifically, co-localization of nascent proteins facilitates interaction between the fusion protein and the Gag precursor polypeptide, thereby increasing the concentration of the POI, such as the RNA guided DNA binding polypeptide described herein, in the nanoparticles provided herein.

Specifically, the Rev response element of HIV-1 is a highly structured, cis-acting RNA element for viral replication that is about 350 nucleotides long. In the wild type HIV virus, it is located in the env coding region of the viral genome and is present on viral mRNA transcripts, serving as an RNA framework onto which multiple molecules of the viral protein Rev assemble. The RRE/Rev oligomeric complex mediates the export of these assemblies from the nucleus to the cytoplasm, where they are translated to produce viral proteins and/or packaged into viral particles.

The RRE is an example of a unique RNA scaffold, providing the framework for assembling a homo-oligomeric complex. The protein-binding sites it presents recruit multiple Rev molecules through diverse sets of interactions with specific positional and orientation requirements. Viral evolution has thus served as a selection experiment, identifying RNA-binding partners for Rev and arranging them structurally to derive maximal functional efficiency from such a complex, even under additional constraints imposed by an overlapping protein-coding reading frame. By deriving specificity from three dimensional restraints imposed by oligomer formation, the virus is able to maintain enhanced specificity of Rev for RRE over the pool of cellular RNAs without relying solely on high-affinity sequence recognition.

According to a specific embodiment, the RRE used herein comprises SEQ ID NO:5, or a functional derivative thereof, preferably comprising at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:5. Specifically, a functional derivative of RRE is capable of promoting nuclear export of a nucleic acid transcript and, as described herein, enhances the packaging of fusion proteins described herein into the nanoparticles described herein.

As described herein, to produce virus-like nanoparticles as described herein comprising a significantly increased number of copies of a heterologous protein, RRE is included in the expression construct comprising the heterologous protein as well as at least one or more expression constructs comprising the gag and/or pol genes.

Specifically, the RRE is located 3’ to the sequence encoding the heterologous protein, 3’ to the gag gene sequence, 3’ to the pol gene sequence, and 3’ or 5’ to the transgene sequence. Specifically, the RRE is located directly after the stop codon in the transcript comprising the sequence encoding the heterologous protein, in the gag transcript and in the pol transcript. Upon translation of the mRNA transcript, a protein is produced that does not comprise the RRE nucleic acid sequence.

Surprisingly, use of the RRE/Rev pathway improves the packaging of RNA guided endonucleases, such as Cas9, into nanoparticles significantly. Specifically, the RRE/Rev promotes cytoplasmic trafficking of e.g. vpr-cas9 transcripts and/or Vpr-Cas9 proteins to the cytoplasmic membrane. Because the gag/pol transcripts also carry RRE, the transcripts and/or Gag proteins are co-localized with the vpr-cas9 transcripts and/or Vpr-Cas9 proteins. The co-localization of Gag and the fusion proteins described herein, such as e.g. Vpr-Cas9, promotes their interaction, which is required for efficient packaging of Vpr-Cas9 into virions.

Specifically, the nanoparticles produced according to the method described herein comprise significantly more copies of the POI (at least 100, 500, 1.000, 5.000, 10.000, or more copies) than nanoparticles produced without the use of an RRE.

The term“nuclear localization signal” or“NILS” as used herein refers to an amino acid sequence, or a nucleotide sequence encoding such AA sequence, that tags a protein for import into the cell nucleus, specifically by nuclear transport. Typically, this signal consists of one or more short sequences, preferably of positively charged lysines or arginines exposed on the protein surface. The amino acid sequence of an NLS specifically comprises about 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids. According to a preferred embodiment, the CRISPR enzyme described herein comprises an N-terminal NLS, preferably the SV40 Large T-antigen comprising the amino acid sequence PKKKRKV (SEQ ID NO:9).

The term "host cell" as referred to herein is understood as any cell type that is susceptible to transformation, transfection, transduction, or the like with nucleic acid constructs or expression vectors comprising polynucleotides encoding expression products described herein, or susceptible to otherwise introduce any or each of the components of the lentivirus-based nanoparticle described herein. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to modifications e.g., by a method described herein, or that occur during replication. Specifically, the host cell described herein is used for the production of the nanoparticle provided herein. According to the method of producing the lentivirus-based nanoparticle described herein, the components of said nanoparticle are introduced into the host cell DNA as polynucleotides encoding said components, which may be stably integrated into the host cell’s genome. Specifically, the host cells are maintained under conditions allowing expression of the components, comprising at least the fusion protein and the gag, pol and env genes as described herein, and optionally one or more gRNA templates, and subsequently allowing formation of the lentivirus-based nanoparticles.

Specifically, the gRNA templates are introduced into the host cell to allow co packaging in the nanoparticle with the CRISPR enzyme. Alternatively, no gRNA templates are introduced into the host cell. Instead, gRNA is introduced directly into the target cell. Examples include but are not limited to, co-transduction of target cells with the CRISPR enzyme-containing nanoparticles and a lentivector carrying the template for gRNA.

Host cells useful for production of the retroviral particles described herein include, e.g., animal cells permissive for the virus, or cells modified so as to be permissive for the virus; or the expression constructs described herein, for example, with the use of a transfection agent such as calcium phosphate. Non-limiting examples of host cell lines useful for producing retroviral particles described herein include, e.g., human embryonic kidney 293 (HEK-293) cells, HEK-293 cells that contain the SV 40 Large T-antigen, human sarcoma cell line HT-1080, glioblastoma-astrocytoma epithelial-like cell line U87- MG, T-lymphoma cell line HuT78, NIH/3T3 cells, Chinese Hamster Ovary cells (CHO), HeLa cells, Vero cells, and the like.

The term “target cell” as used herein refers to any cell type susceptible to penetration and/or integration of the lentivirus-based nanoparticle provided herein. Preferably, the target cell is a mammalian cell and even more preferably a human or rodent cell. The lentivirus-based nanoparticle provided herein is specifically used to modify a genomic sequence in the target cell, which modification can be in vivo, for example in a human being, or in vitro, i.e. in a cell culture. Specifically, the target cell is a somatic cell. Specifically, the target cell comprises a "target nucleotide sequence" which can be any nucleotide sequence of a locus in the nucleic acid of the target cell or population of target cells in which a mutation of at least one nucleotide, such as a mutation of at least one nucleotide in at least one codon (one or more codons), is desired. In particular the one or more gRNAs are designed to target such nucleotide sequence, by a certain degree of complementarity, and where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. Specifically, the degree of complementarity between the guide sequence and the target nucleotide sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm tor aligning sequences. Specifically, the target nucleotide sequence is a non-coding or coding sequence or a combination thereof.

Specifically, the target nucleotide sequence is within a non-transcribed sequence, either upstream or downstream of a coding sequence.

According to a specific aspect, the target nucleotide sequence encodes a protein, preferably an enzyme, a ligand-binding protein, an antibody, a structural protein, a ribozyme, a riboswitch, an RNA, e.g., regulatory RNA, or any other RNA molecule, a group of biomolecules that form a cellular pathway, a regulatory network, a metabolic pathway, a cellular subsystem, or a part of any of the foregoing.

Specifically, the target sequence encodes a therapeutic polypeptide or protein, e.g. a drug-target, a drug-resistance determinant, an efflux-pump, an enzyme, an antigen, a toxin, a ligand-binding protein, an antibiotic-producing gene, a peptide, or a cytokine.

According to a specific embodiment, the nanoparticle described herein is used to deliver a transgene to a target cell. Specifically, the nanoparticle described herein comprises a heterologous transgene, which is to be delivered to a target cell. Advantageously, encapsidating the RNA guided DNA binding polynucleotide described herein as protein instead of as nucleic acid, allows co-packaging of long nucleic acid sequences, such as e.g. therapeutic genes to be delivered to a target cell, in the nanoparticle described herein.

As used herein, the term“transgene” refers to a nucleic acid sequence that is to be delivered to a target cell. Specifically, the transgene is a deoxyribonucleic acid sequence that is delivered to the target cell using the nanoparticles described herein. The transgene may encode a protein, and/or an RNA sequence capable of RNA interference, such as for example pre-miRNA, shRNA or siRNA. Preferably, the transgene is not translated and/or transcribed in the nanoparticle, but is translated into protein, or transcribed into the RNA sequence e.g. pre-miRNA, or shRNA, in the target cell. The transgene may comprise any genetic elements necessary for transient or stable expression of the transgene in the target cell. Specifically, the transgene may be included in the nanoparticle in the form of an expression vector. Preferably, the transgene is placed in the same expression construct as the gRNA template. Thus, according to a preferred example, the transgene is included in the gRNA template vector described herein. With regard to the product encoded by the transgene, there are no limitations. The transgene may encode a protein or a nucleic acid sequence that is to be delivered to the target cell, such as for example an RNA sequence. Specifically, the transgene may encode a therapeutic protein, a reporter protein, an enzyme (e.g. catalase), a pro drug enzyme, an apoptosis inducer, a suicide protein, an anti-immunosuppressive product, an epigenetic modulator, a T cell receptor (TCR), a chimeric antigen receptor (CAR), a protein that modifies the cell surface of transduced cells (e.g.CD52), a protein modifying the expression of the endogenous TCR, a switch receptor that converts pro tumor into anti-tumor signals, a cytokine, or a DNA segment encoding RNA capable of RNA interference, such as pre-miRNA , siRNA or shRNA.

In specific embodiments, the nanoparticle described herein is used to deliver heterologous proteins to a target cell. Specifically, the nanoparticle is used to deliver therapeutic proteins or enzymes, reporter proteins, e.g. fluorescent proteins, pro-drug- activating enzymes, apoptotic proteins, and/or apoptotic enzymes, suicide proteins, cytokines, anti-immunosuppressive products, epigenetic modulators, T cell receptors (TCR), chimeric antigen receptors (CAR), proteins that modify the cell surface of transduced cells (e.g.CD52), proteins modifying the expression of the endogenous TCR, switch receptors that convert pro-tumor into anti-tumor signals. Furthermore, the nanoparticles can deliver DNA segment(s) encoding pre-miRNA(s) or shRNA(s).

According to a specific embodiment, the lentivirus-based nanoparticle provided herein may comprise a surface protein anchored to its membrane, wherein the surface protein has been modified or mutagenized for recognition of a specific target cell. Specifically, the surface protein is a viral surface protein which has been adapted for targeting of a specific cell or population of cells or it is a non-viral surface protein which has been integrated into the membrane of the nanoparticle. The surface protein may be a fusion protein, comprising a viral surface protein or membrane protein fused to a target- specific binding peptide, such as for example an antibody or fragments thereof, or the binding site of any one of an enzyme, an adhesion protein, a ligand or a ligand binding portion of a receptor, which binding site is capable of binding a cognate structure of a binding partner. According to a specific example, the surface protein comprises one or more binding sites of protein domains of antibodies or antibody fragments, or the respective antibody domains or fragments, such as those comprising one, two or more variable antibody domains e.g., Fab, Fv, VH/VL dimer, scFv, dAb, F(ab)2, or other biological binders, such as soluble T-cell receptor, Darpins, etc. As used herein, the term“integrase” refers to an enzyme that enables genetic material to be integrated into DNA of a host or target cell. Specifically, integrase catalyzes the integration of virally derived DNA into the host cell DNA, from which expression products are formed, such as the CRISPR enzyme and the gRNA described herein. Integration can occur at essentially any location in the genome of the host or target cell. Specifically, the integrase may be a viral protein, preferably derived from a retroviral integrase or an engineered, recombinant protein. Preferably, the integrase used herein is an integrase derived from HIV, or a related retrovirus, such as for example Simian immunodeficiency virus (SIV), Bovine immunodeficiency virus (BIV), Caprine arthritis encephalitis virus, Maedi-Visna virus, Avian Sarcoma and Leukosis virus, Moloney Murine Leukemia virus or Prototype Foamy virus. According to a specific example, the integrase described herein is a functional, catalytically active integrase, specifically comprising SEQ ID NO: 10 or a functionally active variant thereof comprising at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:10.

According to a specific embodiment, the integrase is a catalytically inactive integrase. Preferably, the catalytically inactive integrase is used when the nanoparticle described herein does not comprise a transgene. Various methods of rendering an integrase catalytically inactive are known to the person skilled in the art. Preferably, a catalytically inactive integrase is produced by introducing one or more mutations, preferably point mutations, into the amino acid sequence of integrase. Preferably, such mutations are introduced in the catalytic domain of integrase. Exemplary catalytically inactive integrase variants include a D64V mutant of HIV integrase, specifically comprising SEQ ID NO:6 or a variant thereof comprising at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:6. Specifically, the vector used herein is an integration deficient vector. In such case, the gRNA of the CRISPR complex is produced from viral DNA reverse transcribed from the gRNA template described herein. Specifically, using integration deficient vectors comprising a catalytically inactive integrase further increases safety of the delivery system described herein.

The term“polyadenylation signal” or“polyA” as used herein, refers to a stretch of nucleic acid near or at the 3’ terminus of a protein-coding nucleotide sequence comprising a central sequence motif, for example in humans it comprises the amino acid sequence AAUAAA (SEQ ID NO:1 1 ). Specifically, the polyA directs 3’ processing of pre- mRNA to generate mature mRNA.

The term“expression” as used herein regarding expressing a polynucleotide or nucleotide sequence, is meant to encompass at least one step selected from the group consisting of DNA transcription into mRNA, mRNA processing, non-coding mRNA maturation, mRNA export, translation, protein folding and/or protein transport. Nucleic acid molecules containing a desired nucleotide sequence may be used for producing an expression product encoded by such nucleotide sequence e.g., proteins or transcription products such as RNA molecules, in particular fusion proteins or gRNAs as described herein. To express a desired nucleotide sequence, an expression system is conveniently used, which can be an in vitro or in vivo expression system, as necessary to express a certain nucleotide sequence by a host cell or host cell line. Typically, host cells are transfected or transformed with an expression system comprising an expression cassette that comprises the desired nucleotide sequence and a promoter operably linked thereto optionally together with further expression control sequences or other regulatory sequences. Specific expression systems employ expression constructs such as vectors comprising one or more expression cassettes.

The term“expression construct” as used herein, means the vehicle, e.g. vectors or plasmids, by which a DNA sequence is introduced into a host cell so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. “Expression construct” as used herein includes both, autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences.

The terms "vector”,“DNA vector” and "expression vector” mean the vehicle by which a DNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vector” as used herein includes both, autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences, such as artificial chromosomes. Plasmids are preferred vectors of the invention.

Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct.”

A common type of vector is a "plasmid”, which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. Specifically, one or more viral vectors may be used which are selected from the group consisting of lentivirus, retrovirus, adenovirus, adeno- associated virus or herpes simplex virus, lentiviral, adenoviral or adeno-associated viral (AAV) vectors. Specifically, the vectors are selected from the group consisting of HIV-based lentiviruses. Lentiviral vectors may harbor certain safety features, e.g. they may rely on multiple packaging plasmids or they may have truncated long terminal repeats. All of these features are deemed to reduce the chance of obtaining a replication-competent virus, i.e. typically, these viruses can only undergo a single infection cycle.

In specific embodiments, an expression vector may contain more than one expression cassettes, each comprising at least one coding sequence and a promoter in operable linkage.

A "cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. An“expression cassette” as used herein refers to nucleic acid molecules containing a desired coding sequence and control sequences in operable linkage, so that an expression system can use such expression cassette to produce the respective expression products, including e.g., encoded proteins or other expression products. Certain expression systems employ host cells or host cell lines which are transformed or transfected with an expression cassette, which host cells are then capable of producing expression products in vivo. In order to effect transformation of host cells, an expression cassette may be conveniently included in a vector, which is introduced into a host cell; however, the relevant DNA may also be integrated into a host chromosome. A coding sequence is typically a coding DNA or coding DNA sequence which encodes a particular amino acid sequence of a particular polypeptide or protein, or which encodes any other expression product, such as RNA including e.g. the gRNA described herein.

The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Vectors typically comprise DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism. A coding DNA sequence or segment of DNA molecule coding for an expression product can be conveniently inserted into a vector at defined restriction sites. To produce a vector, heterologous foreign DNA can be inserted at one or more restriction sites of a vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. It is preferred that a vector comprises an expression system, e.g. one or more expression cassettes. Expression cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.

To obtain expression, a sequence encoding a desired expression product, such as e.g. any of the polypeptides, proteins or protein domains described herein, or the gRNAs described herein, is typically cloned into an expression vector that contains a promoter to direct transcription. Appropriate expression vectors typically comprise regulatory sequences suitable for expressing coding DNA. Examples of regulatory sequences include promoter, operators, enhancers, ribosomal binding sites, and sequences that control transcription and translation initiation and termination. The regulatory sequences are typically operably linked to the DNA sequence to be expressed.

A promoter is herein understood as a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, one or more nuclear localization signals (NLS) and one or more expression cassettes. Accord ing to a specific embodiment, the gRNA templates described herein are under control or operably linked to an RNA polymerase III promoter. Preferably, RNA pol III promoters, such as U6 and H1 , are used to express these small RNAs.

In some embodiments, one or more vectors driving expression of one or more elements of the CRISPR system described herein and of the lentivirus-based nanoparticle described herein are introduced into a host cell such that expression of the elements of the CRISPR system and the lentivirus-based nanoparticle direct formation of a lentiviral nanoparticle comprising the fusion protein and the gRNA template as described herein.

Expression products such as polypeptides, proteins or protein domains, or RNA molecules as described herein, including e.g., the RNA (gRNA), ribonuclease, fusion proteins, as described herein may be introduced into a host cell either by introducing the respective coding polynucleotide or nucleotide sequence for expressing the expression products within the host cell, or by introducing the respective expression products which are within an expression system or isolated.

Any of the known procedures for introducing expression cassettes, vectors or otherwise introduce (e.g., coding) nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al.).

The term“heterologous” as used herein with respect to a nucleotide sequence, construct such as an expression cassette, amino acid sequence or protein, refers to a compound which is either foreign to a given host cell, i.e.“exogenous”, such as not found in nature in said host cell; or that is naturally found in a given host cell, e.g., is “endogenous”, however, in the context of a heterologous construct or integrated in such heterologous construct, e.g., employing a heterologous nucleic acid fused or in conjunction with an endogenous nucleic acid, thereby rendering the construct heterologous. Specifically, nucleic acid sequences described herein are heterologous. The heterologous nucleotide sequence as found endogenously may also be produced in an unnatural, e.g., greater than expected or greater than naturally found, amount in the cell. The heterologous nucleotide sequence, or a nucleic acid comprising the heterologous nucleotide sequence, possibly differs in sequence from the endogenous nucleotide sequence but encodes the same protein as found endogenously. Specifically, heterologous nucleotide sequences are those not found in the same relationship to a host cell in nature. Any recombinant or artificial nucleotide sequence is understood to be heterologous. An example of a heterologous polynucleotide is an artificial expression cassette as described herein, a nucleotide sequence not natively associated with a promoter, e.g., a heterologous coding sequence with which a promoter is operably linked, such as included in a gRNA vector described herein, or a heterologous transcriptional regulator domain or ribonuclease, described herein, which are foreign to the host cell.

As used herein, the term "mutation" as used herein has its ordinary meaning in the art. A mutation may comprise a point mutation, or refer to areas of sequences, in particular changing contiguous or non-contiguous amino acid sequences. Specifically, a mutation is a point mutation, which is herein understood as a mutation to alter one or more (but only a few) contiguous amino acids, e.g. 1 , or 2, or 3 amino acids are substituted, inserted or deleted at one position in an amino acid sequence. Amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions, as opposed to non-conservative substitutions, comprise substitutions of amino acids belonging to the same set or sub set, such as hydrophobic, polar, etc.

The term "operably linked" as used herein is understood as a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

As used herein, the terms "recognized", "recognizing", or "recognition" in this context refers to the capability of the RNA-guided endonuclease to form a functional complex with a gRNA at a DNA target site which the gRNA hybridizes (i.e. to which the guide sequence of the gRNA hybridizes) in close proximity to a PAM sequence recognized by the RNA-guided ribonuclease described herein and to form a nucleic-acid targeting complex. Specifically, through such complex formation the RNA-guided DNA binding polypeptide is brought into close proximity to, and is thereby directed to the target nucleotide sequence to exert its function.

The term“functional variant” or“functionally active variant” also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, an allelic variant is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or nucleotides or more amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide.

Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence, e.g. by one or more point mutations, wherein the sequence alterations retain or improve a function of the unaltered polypeptide or the nucleotide sequence, when used in combination of the invention. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.

A point mutation is particularly understood as the engineering of a poly-nucleotide that results in the expression of an amino acid sequence that differs from the non- engineered amino acid sequence in the substitution or exchange, deletion or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.

The term“sequence identity” as used herein is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%.

Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50%) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.

Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry. Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different different organisms or species.

To determine the % complementarity of two complementary sequences, one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.

“Percent (%) identity” with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

For purposes described herein, the sequence identity between two amino acid sequences is determined using the NCBI BLAST program version 2.2.29 (Jan-06-2014) with blastp set at the following exemplary parameters: Program: blastp, Word size: 6, Expect value: 10, Hitlist size: 100, Gapcosts: 1 1.1 , Matrix: BLOSUM62, Filter string: F, Genetic Code: 1 , Window Size: 40, Threshold: 21 , Composition-based stats: 2.

"Percent (%) identity" with respect to a nucleotide sequence e.g., of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Optimal alignment may be determined with the use of any suitable algorithm tor aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), EI_AND (lllumina, San Diego, CA), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).

The nanoparticles as described herein may specifically be used in a pharmaceutical composition. Therefore, a pharmaceutical composition is provided which comprise nanoparticles as described herein and a pharmaceutically acceptable carrier or excipient. These pharmaceutical compositions can suitably be administered as a bolus injection or infusion or by continuous infusion. Besides parenteral administration, topic or oral administration may be preferred. Pharmaceutical carriers suitable for facilitating such means of administration are well-known in the art.

Pharmaceutically acceptable carriers generally include any and all suitable solvents, adjuvants, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are physiologically compatible with the lentivirus-based nanoparticle provided herein. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof.

Suitable pharmaceutically acceptable carriers or excipients specifically include one or more of any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, vehicles suitable for topical administration, other antimicrobial agents, isotonic and absorption enhancing or delaying agents, or activity enhancing or delaying agents for pharmaceutically active substances. Common pharmaceutically acceptable additives are disclosed, by way of example, in Remington: the Science & Practice of Pharmacyby Alfonso Gennaro, 20th ed., Lippencott Williams & Wilkins, (2000).

In one embodiment, suitable pharmaceutically acceptable carriers include, but are not limited to, inert solid fillers or diluents and sterile aqueous or organic solutions (e.g., polyethylene glycol, propylene glycol, polyvinyl pyrrolidone, ethanol, benzyl alcohol, etc.). In certain such embodiments, suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, fillers, such as sugars (e.g., lactose, sucrose, mannitol, or sorbitol), and cellulose preparations (e.g., maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone PVP).

In one such aspect, nanoparticles as described herein can be combined with one or more carriers appropriate for a desired route of administration, e.g., admixed with any of lactose, sucrose, starch, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Other carriers, adjuvants, and modes of administration are well known in the pharmaceutical arts. A carrier may include a controlled release material or time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES. Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.

Pharmaceutical compositions are contemplated, wherein a nanoparticle as described herein and one or more therapeutically active agents are formulated. Stable formulations of the lentivirus-based nanoparticle described herein are prepared for storage by mixing said construct having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, in the form of lyophilized formulations or aqueous solutions. Formulations for in vivo administration are preferably sterile, e.g., in the form of a sterile aqueous solution. This is readily accomplished by filtration through sterile filtration membranes or other suitable sterilization methods.

In one embodiment, the pharmaceutical composition comprising a nanoparticle as described herein is administered orally or intravenously. In a specific embodiment, it is administered in a dosage form selected from the group consisting of solid dosage form, a cream, an aqueous mixture or a lyophilized aqueous mixture.

Exemplary formulations as used for parenteral administration include those suitable for subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution, emulsion or suspension. The following items are particular embodiments described herein.

1. A lentivirus-based nanoparticle comprising a fusion protein comprising the following structure from N- to C-terminus:

1. a Viral Protein R (Vpr), or a functional derivative thereof,

ii. optionally a protease cleavage site and/or a linker, and

iii. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof.

2. A lentivirus-based nanoparticle comprising a fusion protein comprising the following structure from N- to C-terminus:

i. a Viral Protein R (Vpr), or a functional derivative thereof,

ii. a protease cleavage site,

iii. optionally a linker, and

iv. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof.

3. The nanoparticle of item 1 or 2, comprising the fusion protein and one or more guide RNA (gRNA) templates comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or single guide RNA (sgRNA).

4. The nanoparticle of any one of items 1 to 3, further comprising a transgene, specifically selected from the group consisting of therapeutic gene, a reporter gene, a gene encoding an enzyme, a gene encoding a pro-drug enzyme, a gene encoding an apoptosis inducer, a gene encoding a fluorescent protein, a gene encoding a pro-drug- activating enzyme, a gene encoding an apoptotic protein, a gene encoding an apoptotic enzyme, a gene encoding a suicide protein, a gene encoding cytokines, a gene encoding an anti-immunosuppressive protein, a gene encoding an epigenetic modulator, a gene encoding a T cell receptor (TCR), a gene encoding a chimeric antigen receptor (CAR), a gene encoding a protein that modifies the cell surface of transduced cells (e.g.CD52), a gene encoding a protein that modifies the expression of the endogenous TCR, and a gene encoding a switch receptor that converts pro-tumor into anti-tumor signals.

5. The nanoparticle of any one of items 1 to 4, further comprising a transgene, wherein the transgene comprises a nucleic acid sequenc encoding an RNA sequence capable of RNA interference, specifically pre-miRNA, siRNA or shRNA.

6. The nanoparticle of any one of items 1 to 5, wherein the RNA guided DNA binding polypeptide is an endonuclease selected from the group consisting of Cas9 and Cpf1. 7. The nanoparticle of any one of items 1 to 6, wherein the fusion protein further comprises a nuclear localization signal (NLS).

8. The nanoparticle of any one of items 1 to 7, wherein the nanoparticle comprises

a) a fusion protein, preferably comprising SEQ ID NO:3 or SEQ ID NO:4, comprising the following structure from N- to C-terminus:

a. a Viral Protein R (Vpr),

b. a protease cleavage site,

c. a linker,

d. a nuclear localization signal (NLS), and

e. a Cas 9 endonuclease; and

b) optionally a transgene; and

c) a gRNA template, which is a sgRNA template.

9. The nanoparticle of any one of items 1 to 7, wherein the RNA guided binding polypeptide is an endonuclease comprising an enzymatically inactive DNAse domain and is fused to a transcriptional regulator domain.

10. The nanoparticle of item 9, wherein the transcriptional regulator domain is a transcriptional activator or a transcriptional repressor.

1 1. The nanoparticle of any one of items 1 to 7, wherein the RNA guided binding polypeptide is an endonuclease fused to an enzyme comprising a deaminase domain, preferably selected from the group consisting of APOBEC1 , ADAR and TadA.

12. The nanoparticle of any one of items 1 to 1 1 , wherein the gRNA template is under control of an RNA polymerase III promoter, preferably a U6 or H1 promoter.

13. The nanoparticle of any one of items 1 to 12, wherein the protease cleavage site of the fusion protein is a human immunodeficiency virus (HIV) protease cleavage site.

14. The nanoparticle of any one of items 1 to 13, wherein Vpr or the functional derivative thereof is derived from a virus belonging to the genus of lentivirus, preferably selected from the group consisting of Human immunodeficiency virus (HIV), specifically HIV-1 or HIV-2, Simian immunodeficiency virus (SIV), Bovine immunodeficiency virus (BIV), Caprine arthritis encephalitis virus, and Maedi-Visna virus.

15. The nanoparticle of any one of items 1 to 14, comprising structural and enzymatic components, wherein the structural components preferably comprise a surface envelope protein, membrane, matrix capsid, nucleocapsid, and p6, and wherein the enzymatic components preferably comprise a reverse transcriptase, a protease and an integrase.

16. Use of the nanoparticles according to any one of items 1 to 15 to modify a genomic DNA sequence in a target cell.

17. The use according to item 16, wherein the target cell is a non-dividing cell.

18. A method of producing the lentivirus-based nanoparticles of any one of items 1 to 15, comprising the following steps of

i. introducing into a host cell the following DNA vectors:

a. an endonuclease vector comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a functional derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably a Cas9 endonuclease;

b. a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA) and an RRE, and optionally a transgene; and

c. one or more vectors comprising a gag gene, a pol gene, an env gene, a rev gene and, optionally, an RRE,

ii. maintaining the host cell under conditions to allow formation of the nanoparticles, and

iii. isolating the nanoparticles.

19. The method of item 18, comprising the following steps of

iv. introducing into a host cell the following DNA vectors:

a. an endonuclease vector comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a functional derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably Cas9 endonuclease,

b. a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA), and a transgene and an RRE, and

c. an envelope vector comprising a nucleic acid sequence encoding an envelope protein, preferably VSV.G,

d. a packaging vector comprising an RRE and a nucleic acid sequence encoding Gag and Pol polyproteins, including an integrase, and

e. a rev vector comprising a nucleic acid sequence encoding a Rev protein, v. maintaining the host cell under conditions to allow formation of the nanoparticles, and

vi. isolating the nanoparticles.

20. The method of items 18 or 19, wherein one or more of the DNA vectors comprise an RNA Polymerase II promoter, preferably one or more of Cytomegalovirus (CMV) promoter, CAG promoter or Rous-Sarcoma-Virus (RSV) promoter.

21. The method of any one of items 18 to 20, wherein the gRNA template vector comprises an RNA Polymerase III promoter, preferably a U6 or H1 promoter.

22. The method of any one of items 18 to 21 , wherein one or more of the vectors comprise a polyadenylation signal.

23. The method of any one of items 18 to 22, wherein the integrase is a catalytically inactive integrase.

24. A kit comprising the following plasmids

i. an endonuclease plasmid comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably Cas9 endonuclease,

ii. an RNA template plasmid comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA) and an RRE, and optionally a transgene,

iii. one or more plasmids comprising a gag gene, a pol gene, an env gene, a rev gene and, optionally, an RRE.

25. A polynucleotide encoding the nanoparticle of any one of items 1 to 15.

26. A cell line comprising the polynucleotide of item 25.

27. A cell line comprising the lentiviral-based nanoparticles of any one of items 1 to 15.

28. A pharmaceutical composition comprising the nanoparticle of any one of items 1 to 15.

29. An expression vector comprising a nucleic acid sequence encoding a fusion protein, which comprises from N- to C-terminus:

a. a Viral Protein R (Vpr)

b. a protease cleavage site

c. optionally a linker, and/or a nuclear localization signal (NLS), and d. an RNA guided DNA binding polynucleotide, preferably an endonuclease;

and a Rev-response element located 3’ to the nucleic acid sequence encoding the fusion protein.

30. The expression vector of item 29, wherein the RNA guided DNA binding polypeptide is Cas9.

31. A method of producing a viral nanoparticle, comprising the steps of introducing into a host cell:

a. a first expression construct encoding a transcript comprising

i. a nucleic acid sequence encoding a viral protein, specifically Viral Protein R (Vpr) or Gag,

ii. optionally a nucleic acid sequence encoding a protease cleavage site and/or a linker between the sequences of a. and c.,

iii. a nucleic acid sequence encoding a heterologous protein, and

iv. at its 3’ end a stop codon followed by a Rev-response element (RRE), and b. one or more second expression constructs comprising a gag gene followed by an RRE, a pol gene followed by an RRE, an env gene, and a rev gene; and

maintaining the host cell under conditions allowing formation of the nanoparticle, followed by isolating the nanoparticle.

32. The method of item 31 , wherein the one or more second expression constructs are selected from the group consisting of

i. an envelope vector comprising a nucleic acid sequence encoding an envelope protein, preferably VSV.G,

ii. a packaging vector comprising an RRE located 3’ to a nucleic acid sequence encoding Gag and GagProPol polyproteins, including an integrase, and

iii. a rev vector comprising a nucleic acid sequence encoding a Rev protein.

33. The method of item 31 or 32, further comprising introducing one or two further expression constructs into the host cell, wherein the one or more further expression constructs are selected from:

a) a gRNA template expression construct comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or guide RNA (gRNA) template, and an RRE,

b) a transgene expression construct comprising a nucleic acid sequence comprising a transgene and an RRE, and c) an expression construct comprising a gRNA template, a transgene and an

RRE.

34. The method of item 31 or 32, wherein the RRE is derived from a lentivirus, preferably selected from the group consisting of human immunodeficiency virus, Simian immunodeficiency virus (SIV), Bovine immunodeficiency virus (BIV), Feline immunodeficiency virus (FIV), Equine infectious anemia virus (EIAV), Caprine arthritis encephalitis virus (CAEV), and Maedi-Visna virus (MVV).

35. The method of any one of items 31 to 34, wherein the RRE comprises SEQ ID NO:5 or a functionally active variant thereof comprising at least 90% sequence identity to SEQ ID NO:5.

36. The method of any one of items 31 to 35, wherein the heterologous protein coding sequence is a sequence encoding a polypeptide selected from the group consisting of DNA binding polypeptides, preferably RNA guided endonucleases, Zinc- Finger Nucleases (ZFN) and Transcription activator-like effector nucleases (TALEN).

37. The method of any one of items 31 to 36, wherein the viral protein is Vpr, or a functionally active variant thereof comprising at least 90% sequence identity to SEQ ID NO:7, and the heterologous protein is a Cas9 endonuclease, or a functionally active variant thereof comprising at least 90% sequence identity to SEQ ID NO:27.

38. The method of any one of items 31 to 37, wherein the heterologous protein coding sequence is a therapeutic gene, a reporter gene, a gene encoding an enzyme, a gene encoding a pro-drug activating enzyme, and/or a gene encoding an apoptosis inducer.

39. A viral nanoparticle produced according to the method of any one of items 31 to 38.

40. The nanoparticle of item 39, for use in the treatment of a disease.

41. Use of the nanoparticle of item 39, to modify a genomic DNA sequence in a target cell in vitro.

42. Use of the nanoparticle of item 39 to modify a genomic DNA sequence in a target cell in vivo in a non-human organism.

43. An expression construct encoding a transcript comprising

i. a nucleic acid sequence encoding Viral Protein R (Vpr),

ii. optionally a nucleic acid sequence encoding a protease cleavage site and/or a linker between the sequences of i. and iii.,

iii. a nucleic acid sequence encoding a heterologous protein, and iv. at its 3’ end a stop codon followed by a Rev-response element (RRE), wherein the RRE directs the transcript to a cytoplasmic microdomain.

44. The expression construct of item 43, comprising from 5’ to 3’ end:

i. a nucleic acid sequence encoding Vpr,

ii. a nucleic acid sequence encoding a protease cleavage site,

iii. a nucleic acid sequence encoding a heterologous protein, and

iv. a stop codon followed by an RRE.

45. A fusion protein expressed by the expression construct of item 43 or 44, wherein the fusion protein comprises

i. Vpr,

ii. optionally, a protease cleavage site and/or a linker between the sequences of i. and iii., and

iii. a heterologous protein.

46. A kit-of-parts comprising

i. the expression construct of item 43 or 44, wherein the expression construct is a plasmid, and

ii. one or more plasmids comprising a gag gene and a pol gene, followed by an RRE and an env gene and a rev gene; and

iii. optionally a plasmid comprising an RRE, as well as a gRNA template and/or a transgene.

The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the spirit and scope of the invention. Accordingly, it should be understood that the examples are illustrative only and are not limiting upon the scope of the invention.

EXAMPLES

Lack of methods for efficient delivery of RNA-guided endonucleases to relevant target cells currently limits the use of gene editing technology in both research and clinical applications. Here, lentivirus-based nanoparticles were engineered to co package Cas9 protein and U6-sgRNA template for their co-delivery to recipient cells. Transduction of un-concentrated, VSV-G envelope-bearing vectors resulted in >98% disruption of the EG FP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, indels formation at a frequency up to 100% and 12% was detected by high- throughput sequencing in targeted endogenous loci in a T cell-derived cell line (SupT1 ) and primary CD4+ T cells, respectively. Thus, the approach represents a novel platform for efficient, safe, and cell-type selective delivery of genome editing enzymes to cells.

Materials and Methods used throughout the Examples:

Cell lines, cell culture, and quantification of EGFP-expressing cells

HEK293T (ATCC CRL-3216), HEK293 cell (ATCC CRL-1573), IM9 (CCL-159), SupT1 (CRL-1942), Jurkat E6-1 (TIB-152), were obtained from ATCC. The THP-1 cells were obtained from Henning Hofmann (Robert Koch Institut). The human kidney cell lines were maintained in stable glutamine-containing high glucose Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10 % fetal bovine serum (FBS Gold Plus, Bio-Sell). The cell lines derived from human lymphocytes and monocytes were cultivated in stable glutamine-containing RPMI-1640 (Carl Roth) supplemented with 10 % FBS Gold Plus (Bio-Sell). Cryopreserved Human CD4+ T cells from normal human peripheral blood were acquired from Zen-Bio. More than 95 % of the cells expressed CD3.

The cells were cultured in X-VIVO 15 (Biozym) + 5 % FBS Gold Plus (Bio-Sell) supplemented with IL-2 (100 ng / ml; PEPROTech) and IL-7 (15 ng /ml; PEPROTech). The cells were activated one day prior to transduction by adding Dynabeads Human T- cell activator CD3/CD28 (Thermo Fisher Scientific) at a bead to cell ratio of 1 :1. For all cultivated cells, no antibiotics were used. The cells were maintained at 37 C and 5 % C02 in a humidified incubator. To detect EGFP-positive cells, UV microscopy with the Olympus IX70 equipped with Olympus XM10 camera and CellSens software (Olympus). 947 ms acquisition time was used to detect the HEK293-EGFP cells expressing a low level of EGFP. To quantify the number of EGFP-positive cells, flow cytometry was performed. The cells were trypsinized, washed and analyzed using FACSCalibour flow cytometer and CellQuest Pro Software (BD Biosciences). Forward versus side scatter gating was used to exclude debris and death cells from the analysis.

Plasmid construction

A pVpr.Prot.Cas9 plasmid was constructed by Gibson assembly of a gBIock ordered from IDT (containing Vpr, Protease cleavage site and SV40 nuclear localization signal-coding sequence) and two PCR products containing Cas9 coding sequence and Rev-responsive element (RRE), respectively. The pLentiCRISPRv2 (available from Addgene; plasmid #52961 ) was used as a template for the amplification of a DNA fragment encompassing the Cas9-coding sequence. The pCMgpRRE plasmid served as a template for the amplification of DNA fragment containing the RRE, CMV promoter, b globin polyA, and plasmid backbone sequences (19).

A Vpr-coding region from HIV-1 YU2 was used as a basis for the design of the gBIock sequence (21 ). A lentiviral transfer vector, Lenti(sgFILLER), which contains a filler sequence flanked by BsmBI sites in place of the 20bp-long targeting crRNA sequence, was constructed from the pLentiCRISPRv2 by deleting Cas9-coding sequence in a long template PCR (primer pair: Fwd: 5’ATG ACC GAG TAC AAG CCC ACG3’ ; Rev: 5’ CCT GTG TTC TGG CGG CAA AC 3’) followed by circularization of the resulting PCR product. Lentiviral transfer vectors carrying sgRNAs targeting specific loci in the genome (Lenti(sgRNA)) were constructed from the Lenti(sgFILLER). The vector was digested with BsmBI and a pair of annealed and phosphorylated oligos was cloned into the single guide scaffold. D64V mutation was introduced into the psPAX2 plasmid by a high-fidelity PCR with a primer pair carrying the desired nucleotide substitution. Plasmids were amplified in DH5a or NEB Stable competent E.coli (New England Biolabs) and purified using a Qiagen Plasmid Midi kit (Qiagen).

Lentivector production and transduction

To produce lentivectors containing both Cas9 protein and U6-sgRNA template five plasmids (a total amount of 3.9 pg) were co-transfected to exponentially growing HEK293T cells seeded in 6-well plates (ATTC CRL-3216; ~80% confluent) using Fugene HD (Promega). We used 1.2 of transfer vector DNA (pLenti(sgRNA)), 0.8 pg of psPAX2 (Addgene #12260) or psPAX2D64V, 0.6 pg pRSV-Rev (Addgene #12253), 0.4 pg of pHCMV-G (20), and 0.9 pg of pVpr.Prot.Cas9.

Transfection medium was replaced with fresh cell culture medium 20 h post transfection. Virus containing supernatants were harvested 48 h post transfection, centrifuged (3500 rpm for 3 min), filtered (Sarstedt), and immediately used. For transduction of adherent cells, ~5 x 10⁴ cells were plated in each well of a 12-well plate one day before transduction. The plated cells were incubated with the virus-containing supernatant (350 pi) supplemented with polybrene (8 pg/ml) for 6 h and fresh cell culture medium (DMEM + 10% FBS Gold Plus)) was added. For transduction of suspension cells, ~1 x 10⁵ cells in 50 pi of cell culture medium were incubated with 100 pi of virus containing supernatant for 6h. Next, cells were pelleted and resuspended in fresh cell culture medium. T7 endonuclease I assay

To detect genomic modification at the targeted regions, genomic DNA was extracted from transduced cells 3 days post-transduction using the Quick Extract DNA Extraction Solution (Lucigen) and used for PCR to amplify specific on-target sites with Phusion high fidelity DNA polymerase (New England Biolabs) and primer pairs specified in Table 1. PCR products were purified by Ampure XP beads (Beckman Coulter) according to the manufacturer’s instructions. 200 ng of purified DNA were denatured and hybridized (95°C, 5min; 95°C to 25°C, -0.1 °C / s; hold at 4°C) in 1x NEBuffer 2 (New England Biolabs) in a total volume of 14mI. 1 mI of T7 Endonuclease I (New England Biolabs) was added to the hybridized PCR product and incubated at 37°C for 30 min. 5 mI of 50 % glycerol was added to the T7 Endonuclease reaction and 20 mI was analyzed on a 2 % agarose gel containing peqGREEN (VWR). The DNA band intensity was quantified using VisionWorks LS Analysis Software. The frequency of indel formation was calculated using the following equation: (1 - (1 - (b + c / a + b + c))1/2 ) x 100, where‘a’ is the band intensity of DNA substrate and‘b’ and‘c’ are the cleavage products (22). It should be noted that we used this equation even though we are aware that it underestimates the editing efficiency in such a case that one type of editing predominates in a highly mutated locus as one mutant DNA duplex upon denaturation and re-annealing produces again mutantmutant hybrid.

High throughput sequencing and data analysis

For deep sequencing, genomic DNA from SupT1 and CD4+ T cells was prepared as described above. The genomic region flanking the targeted site was amplified in 20 cycles using Phusion high fidelity DNA polymerase (New England Biolabs) and the primer pairs specified in Table 1. The amplified sequences were purified (Ampure XP beads (Beckman Coulter)) and send for library preparation and sequencing on a MiSeq high-throughput sequencer (2 x 300 bp; lllumina) to LGC Genomics (Berlin). The 300 bp paired-end MiSeq raw reads were de-multiplexed and low quality reads (a PHRED quality score of less than 30) removed using NextGen Sequence Workbench (Avalanche NextGen). The R1 and R2 fastq files were then uploaded to CRISPR Genome Analyzer (http://crispr-ga.net/) (18) together with the target sequence. Deep sequencing data is available at the NCBI’s Sequencing Read Archive (SRA). Table 1. List of Primers

Example 1 : Construction of lentiviral nanoparticles comprising a Vpr.Prot.Cas9 fusion protein.

It was assumed by the inventors that the large size (~160 kDa) and a net positive charge of the Cas9 might cause structural disturbances leading to reduced transducibility if the endonuclease is directly linked to the structural components of HIV- 1 virions (embedded in Gag polyprotein). Therefore, the inventors translationally fused Cas9 containing an N-terminal protease cleavage site (Prot) to the C-terminus of an accessory HIV-1 protein, Vpr (Vpr.Prot.Cas9; Fig.1 a). Vpr interacts with the p6 domain of Gag precursor, thereby mediating encapsidation of fusion partners into virions. However, relatively few copies of Vpr in viral particles may restrict the applicability of the approach (10). This concern might be especially relevant for intracellular delivery of programmable nucleases that require relatively high amounts for efficient genome editing (9). We speculated that we can address this issue and increase targeting of Cas9 to virions by introducing a potent mRNA nuclear export/cytoplasmic localization functions (Rev-responsive element; RRE) to the Vpr.Prot.Cas9 expression cassette (Fig.1 a). The RRE in combination with an HIV-1 accessory protein Rev (provided in trans), should enhance the cytoplasmic mRNA levels and target the mRNA for translation to the same intracytoplasmic microdomains as the gag mRNAs (also containing RRE) encoding structural and enzymatic components of virions (1 1 , 12). The co-localization of nascent proteins might facilitate interaction between the Vpr.Prot.Cas9 and Gag polyproteins and, in turn, increase the amounts of the nuclease in viral particles.

Figure 1 A shows the design of constructs used to generate the lentivector articles. Cas9 was fused to the C-terminus of Vpr containing an authentic HIV-1 protease cleavage site (CTLNF/PISPI; Vpr.Prot.Cas9). The U6-sgRNA expression cassette was incorporated into a lentiviral expression vector (pLenti(sgRNA)). Packaging construct (psPAX2) encoded either wild-type or inactivated integrase (IN; D64V). VSV.G envelope protein was used to pseudotype and stabilize viral particles (pHCMV-G). Efficient nuclear export and co-localization of mRNA for translation were supported by adding Rev-responsive element (RRE) to the constructs and by overexpressing Rev during virions production (pRSV-Rev). Gag-Pol subunits: matrix (MA), capsid (CA), nucleocapsid (NC), p6, reverse transcriptase (RT), integrase (IN). Packaging signal (y); promoters (CMV, CAG, RSV, U6, EFS), polyadenylation signal (pA), posttranscriptional regulatory element (WPRE).

To produce nuclease-transducing nanoparticles, the fusion-protein-expression construct (pVpr.Prot.Cas9) was co-transfected into HEK293T cells together with four complementary plasmids: i) pHCMV-G, which produces VSV.G envelope protein for pseudotyping of virus particles; ii) psPAX2, a second generation packaging construct, which provides the virion proteins; iii) pRSV-Rev, encoding the Rev; and iv) pLenti(sgRNA) transfer vector containing a U6 promoter driving the expression of a sgRNA specific to the targeted site (Fig. 1 a).

Figure 1 B shows a schematic representation of lentivector-mediated delivery of Cas9 protein and viral RNA containing U6-sgRNA. The Cas9 is packaged into virions as Vpr.Prot.Cas9 fusion polyprotein that is proteolytically cleaved during virion maturation (1 ). Following virus entry to a recipient cell (2), the viral genome is reverse transcribed to DNA (3) and together with the Cas9 is translocated to the nucleus (4), where the U6 promoter drives the expression of sgRNA (5). The nascent sgRNA associates with Cas9

(6) and directs the nuclease to the target site in the genomic DNA (gDNA) for cleavage

(7).

Example 2: Characterization of the lentiviral nanoparticles comprising a Vpr.Prot.Cas9 fusion protein and a sgRNA template.

An EGFP disruption assay was used to determine the ability of the“bi-component” lentiviral vector for combined transduction of Cas9 protein and U6-sgRNA expression cassette (hereon referred to as VECTR), produced from the transfected cells, to deliver Cas9 protein to the nucleus of mammalian cells, form a complex with a nascent sgRNA and induce mutagenesis. A specific site in a single copy of the EGFP reporter gene incorporated in the chromosome 17 of the genome of HEK293 cells was targeted (HEK293-EGFP; Fig.2A). Remarkably, transduction of un-concentrated VECTR(sgGFP) resulted in a robust loss of EGFP in ~90% of the EGFP-expressing cells (Fig. 2B). The disruption activity was almost as high as that obtained with a potent cas9 gene-delivering lentiviral vector pLentiCRISPRv2(aGFP) (13). The EGFP expression remained unchanged following transduction of a control lentivector either lacking Vpr.Prot.Cas9 protein or containing sgRNA designed to target an EMX1 locus (Fig.2B). Furthermore, no significant EGFP disruption was observed when the vector particles were prepared in the absence of VSV.G envelope protein or when an inhibitor of reverse transcription, azidothymidine (AZT), was added to transduced cells (Fig.2B).

Figure 2A shows the integration site as determined by Sanger sequencing of the LM-PCR product (linker sequence is shown in bold, human sequence in italics and vector sequence is underlined). Figure 2B shows EGFP gene disruption in HEK293- EGFP cells after transduction with the bi-component lentivectors (black entries) or a control LentiCRISPRv2(sgGFP) (white entry).

To confirm that the marked disruption of EGFP arose from genome modification and not from Cas9 binding or cellular toxicity, a T7 endonuclease (T7E1 ) assay and an Inference of CRISPR Edits (ICE) analysis (ice.synthego.com) was used to detect and quantify the frequency of insertions/deletions (indels) at the target EGFP locus. Both assays verified the presence of VECTR-induced double-strand-breaks (DSBs) corrected by the error-prone non-homologous end joining (NHEJ), although the gene disruption frequencies somewhat varied (38% for T7E1 vs. 97% for ICE) (Fig. 3 and Fig. 4). The difference is perhaps due to a high frequency of +1 nt insertions (37% of indels) that increased the likelihood of re-annealing of the mutant DNA strands leading to the insensitivity of the resulting homoduplexes to T7E1 (Fig.3B) (14).

Figure 3A shows a T7 endonuclease I (T7EI) assay to measure indels in EGFP gene resulting from transductions with the two-component lentivector, the same vector lacking Vpr.Prot.Cas9 or the control pLentiCRISPRv2(sgGFP) and Figure 3B shows mutant sequences at the EGFP locus and their frequencies as determined by SYNTHEGO analysis of Sanger sequencing of a PCR product amplified from VECTR(sgGFP)-transduced HEK293-EGFP cells. The 20-nt target sequence is shown with a grey background. The PAM sequence is shown in bold.

Figure 4 shows determination of EGFP gene editing efficiency by Inference of CRISPR Edits (ICE from SYNTHEGO). An amplicon obtained from HEK293-EGFP cells transduced with bi-component VECTR(sgGFP) was analyzed by ICE. PCR product from mock-transduced cells was used as a control. Additional data to Fig. 3B (A) Summary of editing results. (B) Sanger sequencing chromatograms of the edited (upper panel) and control (lower panel) samples. The horizontal black underlined region represents the guide region. The PAM is underlined in red. The cut site is shown by the vertical dotted line. (C) The left panel shows the level of disagreement between the control and edited samples around the cut site. Distribution of indel sizes and their frequencies is depicted in the right panel.

Following transduction, no appreciable toxicity was observed in HEK293-EGFP cells and this observation was also validated by an XTT assay that showed only a minimal loss of cell viability after the transduction (Fig. 5 A and B).

Figure 5 shows transduction with two-component lentivector has minimal effect on cell viability. (A) Cell viability was measured by the Cell Proliferation (XTT) assay (Roche) three days post transduction with VECTRv2(sgGFP). HEK293-EGFP cells transduced in 12 well plates were incubated with yellow XTT solution (final concentration 0.3 mg/ml; 2 h) added directly to the cell culture media. Next, formation of orange formazan dye in cell culture medium by viable cells was quantified by an ELISA reader. As controls mock transduced cells, cells co-transfected with Lenti(sgGFP) + pVpr.Prot.Cas9 plasmids (Fugene HD; Promega) or cells treated with hydroxyurea (HU; 0.2 or 0.5 mM) were used. Mean viabilities of two biological replicates are shown. Error bars, mean ± SD. (B) Quantification of formazan dye formed from XTT tetrazolium salt added to different amounts of cells seeded in 12 well plates. Mean absorbance values ± SD from two replicates are shown. (C) Titration of the pVpr.Prot.Cas9 plasmid for optimal gene disruption activity. HEK293T cells were transfected with a total amount of 4 pg of plasmid DNA. The amount of the Cas9 expression construct varied from 0 pg to 1.2 pg (adjusted to 1.2 pg by “empty” pcDNA3). As a positive control LentiCRISPRv2(sgGFP) prepared in the absence of pVpr.Prot.Cas9 was used. Error bars reflect SD from three replicates.

Next, the amount of pVpr.Prot.Cas9 plasmid used for the VECTR(sgGFP) preparation was titrated and it was found that the highest extent of the reporter EGFP gene disruption (~98%) was achieved with 0.9 pg of the construct in a total amount of 4 pg of DNA used for transfection (Fig. 5C).

Example 3: Lentiviral nanoparticles comprising a catalytically inactive integrase.

Only a short boost of activity is needed for efficient knockouts mediated by programmable nucleases, therefore we hypothesized that the expression of sgRNAs from unintegrated viral DNA might be sufficient for gene ablation. Further, as the integration of lentiviral DNA is an undesired side effect, the use of an integration deficient vector would improve the safety of two-component nanoparticles. To explore whether an integration-deficient lentiviral vector can be used for an efficient gene knockout, vector particles containing a catalytically inactive integrase protein (D64V, VECTRv2(sgGFP)) (15) were produced and tested for their EGFP disruption activity.

In direct comparison with integration proficient VECTR(sgGFP), it was found that there was no detectable loss of the gene ablation activity, indicating that the use of the integration deficient vector is a viable modification of the system (Fig. 6).

Figure 6A shows comparison of EGFP disruption after transduction with lentiviral particles containing integration deficient (D64V) or proficient (WT) integrase. Figure 6B shows Integration deficient vector mediates EGFP disruption as efficiently as the vector containing wild-type (WT) integrase (IN). The expression of sgRNA from episomal non- integrated viral DNA forms is sufficient for a high level gene disruption. For this experiment viral vectors were prepared with either psPAX2 (WT IN) or psPAX2D64V (D64V IN) packaging construct. The psPAX2D64V encodes an integrase with the D64V mutation in the active center that abolishes integration activity of the enzyme. Representative examples of flow cytometry dot blots from three replicates are shown. LentiCRISPRv2(sgGFP) containing WT IN (encoded from psPAX2 plasmid) served as positive control. The percentage of EGFP positive cells is shown in the upper right corner.

Example 4: Comparison of delivery of Cas9 as a protein versus delivery as a gene.

To examine dose-response, HEK293-EGFP cells were transduced with decreasing amounts of lentiviral vectors. A positive correlation between lentiviral vector dose and the EGFP disruption activity was observed. The loss of knockout activity was more pronounced for the bi-component VECTRv2(sgGFP) nanoparticles compared to the gene-delivering pLentiCRISPRv2(sgGFP), consistent with a prediction that direct protein delivery is more vulnerable to loss of effective nuclease protein concentrations compared to the administration of nuclease gene expression cassette.

To compare the kinetics of EGFP disruption between VECTRv2(sgGFP) and pLentiCRISPRv2(sgGFP), the expression of EGFP was followed over an 80 h period after the transduction. No difference in the time course of gene disruption was detetcted. Both RGEN delivery methods required a lag period over 24 h to show an EGFP disruption activity followed by a steady increase, which plateaued at 56 h post transduction (Fig. 7 A and B). Additionally, we observed that the loss of fluorescence remained stable over a period of 35 days after the treatment (Fig. 1C). These results, combined with the findings with AZT, which indicated that reverse transcription is required for site-specific mutagenesis (Fig.2b), support a model whereby the formation of a complex between Cas9 and sgRNA takes place in the nucleus of the recipient cells rather than during the assembly of viral particles in the transfected cells (Fig.1 B).

Figure 7 shows time course of EGFP knockout after transduction with the bi component Cas9 protein-containing VECTRv2 or cas9 gene-carrying lentivector (LentiCRISPRv2(sgGFP)). Figure 7B shows that flow cytometry was used to determine the percentage of EGFP-expressing cells at various time points after transduction that was then used for the calculation of disruption activity in Figure 7A. Dot blots of one of two replicates are shown. The percentage of EGFP positive cells is shown in the upper right corner. Figure 7C shows a long term EGFP disruption experiment. EGFP expression in HEK293EGFP cells transduced with EGFP- or EMX1 -targeting bi component VECTRv2 was measured over a period of 35 days post transduction. The EGFP disruption values reflect means ± SD from four replicates.

Given these results, it was next investigated whether the VECTRv2 system compared to the lentivirus-mediated administration of nucleic acids is more sensitive to Watson-Crick mismatches at the sgRNA-DNA interface. For this purpose, we determined EGFP disruption activity for both lentivirus-based delivery systems bearing variants of the original sgRNA (sgGFP site no.1 , positions 1 -19 in bold of SEQ ID NO:2, Fig. 8A) with adjacent double mismatches at positions 1 -19 (numbered in the 3’ to 5’ direction). Regardless of RGEN administration route, a robust EGFP disruption activity, equivalent to that mediated by the matched sgRNA, was observed for the sgRNA containing mismatches at positions 17&18 and 18&19, respectively. The sgRNA containing mismatches at position 15&16 induced less potent editing relative to the matched sgRNA and the loss of activity was more dramatic when the sgRNA was transduced into cells together with Cas9 protein. Of the remaining seven sgRNAs, only the sgGFP (1 1&12) showed an appreciable activity, but only when delivered via the pLentiCRISPRv2 encoding the nuclease (Fig.8B, C and D). Collectively, these results confirm previous reports that mismatches at the 5’end of sgRNAs are better tolerated than those at the 3’ end and establish that the VECTR-mediated editing is more specific compared to the mutagenesis induced by transductions of cas9 gene into cells (16,17). Figure 8 shows EGFP disruption activity of Cas9 protein- and cas9 gene-carrying lentivectors containing either matched sgRNA (site 1 ) or sgRNA bearing double adjacent mismatches at the indicated positions. Figure 8A shows the targeted EGFP sequence. Figure 8B shows the EGFP gene disruption in HEK293-EGFP cells after transduction with the bi-component lentivectors (black entries) or a control LentiCRISPRv2(sgGFP) (white entry). The gRNAs contained mismatched dinucleotides. The gRNA with sequence matching the target locus was used as a control. Figures 8 C and D show flow cytometry dot blots reflecting representative examples of three replicates used to calculate mean ± SD EGFP disruption activity shown in Fig 8B. The percentage of EGFP positive cells is shown in the upper right corner of the dot blots.

Example 5: Modification of endogenous genes using the lentiviral nanoparticles.

Next, it was tested whether virions delivering Cas9 protei template sgRNA can induce cleavage of endogenous genes in human HEK293-GFP cells. Transductions of VECTRv2 targeted to the EMX1 , FANCF, HEKsitel and HEKsite3 loci in the human genome resulted in indel formation in all four genomic loci with efficiencies similar to those achieved with cas9 gene-delivering virions (13%— 55%), as revealed by T7E1 assay (Fig. 9A).

Having determined on-target mutagenesis of endogenous loci in HEK293 cell, next we sought to assess whether the VECTRv2 can induce site-specific DSBs in technically more challenging cell types including Jurkat, SupT1 , IM9, and THP-1 cell lines as well as in primary CD4+ T cells. A formation of indels at variable frequencies ranging from 6% to 70% was detected in all the cell types (Fig. 9B and C). Whereas IM9, THP-1 , and CD4+ T cells were less edited, the two T cell-derived lines, Jurkat and SupT1 , were comparably sensitive to the induction of mutations as HEK293-GFP cells. Amplicons generated using genomic DNA from SupT 1 and CD4+ T cells were then used for high-throughput sequencing to verify and precisely quantify on-target mutations at the FANCF, HEKsl , and HEKs3 loci. For CD4+ cells, we found indels at a frequency of 2%, 1 1 %, and 12%, respectively, and transduction of SupT1 yielded cleavage efficiencies of 70%, 100% and 100%, respectively (Fig. 9D and E).

Figure 9 shows RNA-guided genome editing of the native loci in multiple cell types. Figure 9A shows detection of indels by T7E1 assay performed on endogenous EMX1 , FANCF, HEKsl , HEKs3 loci in HEK293-EGFP cells transduced with VECTRv2(sgRNA) or LentiCRISPRv2(sgRNA). Figure 9B shows VECTRv2(sgRNA)- mediated mutations on FANCF, HEKsl and HEKs3 loci in lines of human T (Jurkat, SupT 1 ) and B (IM9) lymphocytes, and monocytic cell line (THP-1 ) as measured by T7E1 assay. Mutation rates obtained from parallel transductions of HEK293-EGFP cells are also shown. Figure 9C shows NHEJ rates (measured by T7E1 assay) in primary CD4+ T cells transduced with VECTRv2(sgRNA). Parallel transductions of SupT1 cells served as positive control. Please note that the HEKs3 site could not be analyzed by T7E1 assay due to an SNP near the cut site. Figures 9D-E shows NHEJ frequencies as quantified by CRISPR Genome Analyzer using next-generation sequencing data of amplicons from (C) as an input (18), (D) SupT1 T cell line; (E) primary CD4+ T cells.

Example 6: Comparison of Cas9 fused to Vpr versus Cas9 fused to Gag.

To find out if Cas9 can be packaged into lentiviral nanoparticles as fusion with the Gag polyprotein, we fused Cas9 protein with the matrix (MA) subunit of the Gag polypeptide. The resulting plasmid pGag.Cas9 (0 pg, 0.3 pg, 0.6 pg, and 0.9 pg) was co-transfected with psPAX2 (0.8 pg), pLenti(sgGFP) (1.2 pg) pRSV-Rev (0.6 pg), pHCMV-G (0.4 pg), and pcDNA3 (to adjust total DNA amount to 3.9 pg) into 293T cells. As a positive control, nanoparticles containing Vpr.Prot.Cas9 fusion protein were prepared in parallel co-transfections. Viral particles released from the transfected cells were used to transduce HEK293-EGFP cells. Transduction of un-concentrated Gag.Cas9 fusion protein-containing lentivectors resulted in disruption the GFP gene in up to ~48 % (with 0.6 pg of pGag.Cas9 plasmid) of targeted cells (Figure 10). Surprisingly, transductions with lentivectors carrying Vpr.Prot.Cas9 polyprotein showed a significantly greater EGFP disruption activity ranging from ~80 % (0.3 pg of pVpr.Prot.Cas9) to ~97 % (0.9 pg of pVpr.Prot.Cas9). Based on these data we conclude that Cas9 protein can be delivered to target cells as fusion with Gag polyprotein. However, the Cas9 nuclease delivered to cells by these means is less active when compared with Cas9 packaged into lentivector particles as Vpr.Prot.Cas9 fusion protein.

Figure 10 shows EGFP disruption activity of lentivectors carrying either Gag.Cas9 or Vpr.Pro.Cas9 fusion protein. The lentivectors were prepared by transient transfection of HEK293T cells. The amount of the Cas9-encoding plasmid (Vpr.Prot.Cas9 or Gag.Cas9) was titrated. Total DNA content was adjusted by pcDNA3 to 4pg. Lentivector for the expression of Cas9 in target cells LentiCRISPRv2(sgGFP) was used as a positive control. Error bars represent SD from three replicates. Conclusions

Herein, a unique concept of co-delivery of Cas9 protein and a template for sgRNA within lentivirus-based “nanoparticles” is shown. The concept is built upon earlier findings that lentiviruses can be exploited as multicomponent tools for simultaneous delivery of foreign proteins and an episomal viral DNA, which is generated by reverse transcription from the vector RNA genome (8,9). Herein, it was surprisingly demonstrated that the episomal DNA can further serve as a template for transcription of sgRNA, which then forms a complex with the co-delivered Cas9 protein and targets the nuclease to a specific site in the genome.

Remarkably, this strategy led to a robust editing activity that was comparable or even superior to that reported for direct delivery of Cas9 protein/sgRNA complexes to cells (1 ,2). In contrast to the chemical transfection or electroporation, the virus-mediated delivery is receptor-mediated and hence it allows selective transfer to essentially any target cell population by using pseudotypes bearing various natural or engineered envelope proteins (6). Furthermore, the use of this approach may extend the repertoire of cells types that could be edited. Importantly, this includes clinically relevant non dividing cells (neurons, hepatocytes, quiescent lymphocytes, and hematopoietic stem cells, etc.) as they are permissive for the lentivector-mediated transduction of cargos to the nucleus (5).

Several factors contribute to the high efficiency of the approach. First, Cas9 is fused to Vpr rather than to Gag polyprotein. Incorporation of the bulky nuclease to Gag causes structural instability of virions resulting in reduced efficiency. Second, incorporation of an RRE element to the expression constructs, including the packaging and pVpr.Prot.Cas9 plasmids facilitated nuclear export of the respective mRNAs and directed them to the same cytoplasmic location for translation. Subsequently, co localization of the nascent proteins might facilitate interaction between p6 and Vpr.Prot.Cas9 and, in turn, incorporation of the fusion protein into virions. Third, after receptor-facilitated cell entry, Cas9 protein remains embedded in a lentiviral capsid (protected from degradation) and exploits the intracellular trafficking routes to reach the chromosomes. Fourth, coordinated intra-nuclear delivery of Cas9 and viral DNA as part of a pre-integration complex places the nuclease in close proximity to nascent sgRNA molecules and facilitate RNP complex formation (Fig.1 B).

In summary, multi-component lentiviral nanoparticles ferrying Cas9 protei sgRNA template to the nuclei of transduced cells for transient exposure of the genome to the nuclease allowing specific disruption of targeted genes are described. The presented system represents a versatile platform for efficient, safe, non-toxic and cell-type selective delivery of genome modification enzymes to cells.

Example 7: Enhancement of the production of lentiviral nanoparticles comprising Cas9 nuclease.

The packaging construct psPAX2 used in Example 1 , in which the expression of viral genes is driven by a CAGG promoter, is replaced with a new construct containing CMV or EF1 a promoter. These promoters are cloned in place of the CAGG promoter. The resulting packaging plasmids are co-transfected with a Tax expression construct into HEK293T cells and the production of lentiviral nanoparticles is compared with that obtained using psPAX2. Following initial testing with a lentiviral vector carrying the EGFP gene showing enhanced infectivity, the new packaging construct is used for enhanced production of lentiviral nanoparticles containing Cas9 nuclease and a template for sgRNA.

Example 8: Efficiency of the packaging of viral nanoparticles with and without RRE.

Previous reports with directed endonucleases such as meganucleases, packaged to lentiviral particles with the help of Vpr, showed only a limited DNA cutting efficiency (8). A moderate genome editing efficiency was also reported for other directed nucleases such as zinc finger nucleases (ZFN) and TALEN nucleases, packaged into virions with the help of the viral structural protein, Gag (9). The low efficiency resulted in limited usage of the lentiviral nanoparticles carrying the nuclease protein. In fact, the lack of efficient and safe transient nuclease delivery methods can be considered as one of the reasons for the departure from programs based on the employment of meganucleases.

At least two reasons are responsible for low genome editing efficiency using viral nanoparticles.

1. First, and probably the most important reason: Lentiviral virions contain a protease that degrades foreign protein packaged into viral particles. One strategy to avoid proteolytic cleavage would be to identify protease cleavage sites in the packaged protein and to eliminate these sites. However, this is laborious and not possible for many proteins. 2. The second reason for observed moderate gene ablation activity with Vpr- containing fusion protein is that the Vpr is a low copy-number viral protein. It has been calculated that viral particles contain only a few hundred Vpr copies. This strongly contrasts with the amount of viral structural subunit proteins (Gag). More than 5000 copies of Gag are present in virions (10). Therefore, many attempts to package foreign proteins focused on the fusion of heterologous proteins with Gag (7, 23, 24, 25). The fusions with Gag result in a greater amount of the foreign protein in virions as compared to the fusion protein containing Vpr. Therefore, it is expected that it will improve the titration of viral protease activity and increase genome editing efficiency. However, fusions with Gag lead to misfolding of the fusion proteins, structural disturbances and ultimately to reduced infectivity of viral particles. This effect is salient for bulky proteins including directed nucleases (e.g. Cas9 has a molecular mass exceeding 160 kDa).

In order to overcome these problems, it was tested whether usage of the accessory protein Vpr would preserve viral infectivity and at the same time would package high numbers of fusion protein to the nanoparticle to enable efficient DNA modification in target cells. It was postulated that encapsidating a high amount of fusion protein in the nanoparticle could titrate out the viral protease activity.

Design of the constructs to generate the lentivector particles is shown in Figure 19A. Cas9 was fused to the C-terminus of Vpr containing an authentic HIV-1 protease cleavage site (CTLNF/PISPI; Vpr.Prot.Cas9). The U6-sgRNA expression cassette was incorporated into a lentiviral expression vector (Lenti(sgRNA)). The packaging construct (psPAX2) encodes the structural and enzymatic components of virions. The VSV.G envelope protein was used to pseudotype and stabilize viral particles (pHCMV-G). Efficient nuclear export and colocalization of mRNA for translation were supported by adding the Rev-responsive element (RRE) to the constructs and by overexpressing Rev during virion production (pRSV-Rev). Gag-Pol subunits: matrix (MA), capsid (CA), nucleocapsid (NC), p6, reverse transcriptase (RT), and integrase (IN). Packaging signal (y); promoters (CMV, CAG, RSV, U6, and EFS), polyadenylation signal (pA), post- transcriptional regulatory element (WPRE). When no RRE was present in the expression cassette, only a moderate EGFP ablation efficiency (~25%) was observed with Vpr.Prot.Cas9 fusion protein (Lenti(sgGFP)+Vpr.Prot.Cas9) (Figure 19B).

To test the postulation, that the amount of encapsidated fusion protein could be increased by controlling the localization of mRNA encoding the fusion protein, the rev responsive element (RRE) was appended to the mRNA encoding the Vpr-Cas9 fusion protein. The RRE is also present in the mRNA encoding Gag. Design of the constructs to generate the lentivector particles is shown in Figure 20A.

As shown in Figure 20B, the introduction of RRE resulted in a marked improvement of gene editing activity. The unconcentrated virus preparations carrying Vpr.Prot.Cas9 ablated EGFP gene in ~92% of target cells.

This shows that the identical spatiotemporal control of the posttranscriptional processes of mRNA encoding the structural components of virions and the fusion proteins results in the co-localization of the mRNAs in a cytoplasmic microdomain for translation. Co-localization of the translation products - the nascent Gag and Vpr-Cas9 proteins - promotes their interaction, which is a pre-requisite for the packaging of Vpr- Cas9 into virions. Enhanced packaging titrates out the protease activity and promotes the efficient ablation of target genes.

Conclusion:

Fusion of Cas9 to Vpr alone is not sufficient to achieve high levels of intra-viral Cas9 protein and, in turn, high genome editing potency. The presence of RRE in the expression cassette fixes this insufficiency and mediates the packaging of high amounts of Cas9 proteins into virions, resulting in high-level EGFP disruption. The improvement of the packaging efficiency permits usage of unconcentrated viral preparations, while prior art viral particles had to be concentrated, e.g. by ultracentrifugation, which made the production of such particles laborious.

Example 9: Efficiency of the RRE-Nuclear Export System

To investigate the effect of nuclear export system on the expression and the packaging ofVpr.Prot.Cas9, two Vpr.Prot.Cas9 expression constructs, pVpr.Prot.Cas9- CTEsx and pVpr.Prot.Cas9-RRE (Fig.12), were generated. The pVpr.Prot.Cas9-CTE3x contains three copies of a constitutive transport element (CTE) derived from Mason Pfizer Virus (MPMV) that facilitate nuclear export of RNAs via the NXF1 pathway. The pVpr.Prot.Cas9-RRE contains RRE from HIV-1 , which directs the nuclear exit of mRNA to the CRM1 pathway. To express Vpr.Prot.Cas9 and package the fusion protein into lentiviral nanoparticles, the expression plasmids were transfected to HEK293T cells together with four complementary plasmids: i) pHCMV-G, which produces VSV.G envelope protein for pseudotyping of virus particles; ii) pLenti(sgGFP)-puro transfer vector; iii) pRSV-Rev (for pVpr.Prot.Cas9-RRE) or“empty” pcDNA3 (for pVpr.Prot.Cas9- CTEsx), and iv) psPAX2, a second generation packaging construct that provides the virion proteins. Importantly, the gag/pro/pol mRNA derived from the psPAX2 plasmid contains RRE, therefore the nuclear export of the mRNA occurs via the CRM1 pathway (Fig.12).

The vector particles were harvested from the cell culture supernatants of transfected cells two days post-transfection and concentrated by ultracentrifugation (100 fold). Additionally, cell lysates were prepared from the same cells. Viral and cellular proteins were resolved on a PAGE gel, transferred onto a PVDF membrane and probed with an antibody specific to Cas9 (Santa Cruz). As shown in Fig.13 (upper blot), the Vpr.Prot.Cas9 expression levels detected in cell lysates were virtually identical for both constructs, pVpr.Prot.Cas9-CTE3x (CTE3x) and pVpr.Prot.Cas9-RRE (RRE).

This result strongly contrasted with the data obtained for the levels of Vpr.Prot.Cas9 proteins in concentrated virus preparations. While Vpr.Prot.Cas9 protein expressed from the RRE-containing transcripts were readily detectable, no proteins could be detected when the nuclease was expressed from the CTE-containing transcripts (Fig.13, upper blot). This result was not due to a differential loading or blotting of viral proteins as the same levels of Gag (Pr55, p24) were detected when the membrane was re-probed with anti-p24 antibody (Fig.13, middle blot). The same loading of cellular lysates was confirmed by using antibody specific to HSP90 protein (Fig.13, lower blot) and also by Coomassie blue staining of the gel after blotting (Fig.13, Coomassie blue stained gel).

Taken together, these data demonstrate that nuclear export of vpr-cas9 transcripts via the CRM1 pathway (RRE/Rev) facilitates packaging of Vpr.Prot.Cas9 proteins into lentivector nanoparticles in the VECTR-Cas system. These results are consistent with the model (see Fig. 14) that the RRE/Rev promotes cytoplasmic trafficking of vpr-cas9 transcripts and/or Vpr.Prot.Cas9 proteins to the cytoplasmic membrane, where the assembly of lentiviral particles occurs. Because the gag/pro/pol transcripts also carry RRE, the transcripts and/or nascent Gag proteins co-localize with vpr-cas9 transcripts and/or Vpr.Prot.Cas9 protein. The co-localization of Gag and Vpr.Prot.Cas9 promotes their interaction, which is required for packaging of Vpr.Prot.Cas9 into virions (see schematic representation in Fig. 14).

The vpr-cas9 transcript and/or Vpr.Prot.Cas9 proteins expressed from transcripts containing CTE (or no export element) are not localized to the cytoplasmic membrane (remain in the peri-nuclear region). As a result, the interaction between Vpr.Prot.Cas9 and Gag is hindered resulting in an inefficient packaging of Vpr.Prot.Cas9. This is the first demonstration that the nuclear export pathway has an effect on the packaging of heterologous (non-viral) proteins. The same approach can be used to promote high- efficiency packaging of other heterologous proteins, including ZFN and TALEN nucleases.

Example 10: Use of the VECTR-Cas system for simultaneous gene disruption and delivery of a transgene to target cells

The experiment outlined here demonstrates that the VECTR-Cas lentiviral nanoparticles can be modified to simultaneously knock-out a gene of interest (e.g .EGFP) and deliver a transgene of interest (e.g. a red fluorescent protein ( DsRed ), a chimeric antigen receptor (CAR)) to target cells.

In the previous examples, it was demonstrated that the Cas9 protein is efficiently packaged into lentiviral nanoparticles when fused to the lentiviral accessory protein (Vpr) and when expressed from transcripts containing the Rev responsive element (RRE). The packaged protein is efficiently delivered to the nuclei of transduced cells and forms a complex with a nascent sgRNA. The complex is targeted to the host DNA and disrupts the targeted gene in the host genome.

In contrast to other lentiviral Cas9 delivery systems, which deliver the cas9 gene, the VECTR-Cas system directly delivers the Cas9 protein to target cells. The lack of cas9 gene in the VECTR-Cas system allowed to insert another transgene into the lentiviral nanoparticles (see Fig. 15). The simultaneous delivery of the cas9 gene and a transgene of interest in previous models was impossible due to the size limit of inserts in the lentivector genome. The lentivector genome of the present nanoparticles comprises the sgRNA template to guide the Cas9 protein to the target site of the target cell’s genome, but it does no comprise the cas9 gene. To demonstrate that it is possible to concurrently ablate a gene of interest and deliver a transgene to target cells, the VECTR-Cas nanoparticles were used containing 1 ) the Cas9 protein, 2) a template for sgRNA targeting the EGFP gene (sgGFP) and 3) a transgene for the expression of a red fluorescent protein (DsRed). The transgene together with a promoter (EF1 alpha) was cloned into the lentiviral transfer vector pLenti(sgGFP) downstream of the U6-sgRNA transcription unit. To produce the VECTR- Cas(sgGFP -DsRed) lentiviral nanoparticles, the transfer vector was co-transfected into 293T cells together with Vpr.Prot.Cas9 expression construct, psPAX2 packaging construct, and with pRSV-Rev and pHCMV-G plasmids (see Fig. 16). Lentiviral nanoparticles were collected two days after transfection and used for the transduction of target cells expressing EGFP protein, 293GFP.

The target cells were inspected three days after transduction for the expression of the green (EGFP) and red (DsRed) fluorescent proteins.

Figure 17 shows the simultaneous knock-out of an endogenous gene (eg/p) and delivery of a transgene (DsRed). HEK293GFP cells carrying a single copy of egfp gene were transduced with three-component lentivector nanoparticles VECTRv2- Cas(sgGFP)-DsRed containing the Cas9 protein, a template for sgRNA targeting the egfp gene (sgGFP), and the DsRed transgene under the control of EF1 a promoter. As a control the cells were transduced with control lentiviral nanoparticles (VECTRv2- Cas(sgEMX)-puro). The EGFP and DsRed expression was monitored by flow cytometry and UV microscopy three days post transduction. The expression of EGFP was disrupted in >80% of HEK293EGFP cells transduced with VECTRv2-Cas(sgGFP)- DsRed. Simultaneously, 99% of the transduced cells expressed DsRed (left figures). In contrast, transduction with the control lentivector did not result in a reduction of the EGFP expression (98% of cells produced EGFP) (right figures).

Specifically, Figure 17 shows that transduction with the VECTR-Cas(sgGFP- DsRed) lentivectors resulted in a marked loss of the EGFP expression in ~90% of 293GFP cells. Concurrently, approximately 99% of the targeted cells expressed the DsRed.

This“proof-of-concept” result demonstrates that the VECTR-Cas(sgGFP -DsRed) lentivectors can simultaneously knock-out the EGFP gene and deliver DsRed transgene to 293GFP cells. Next, it was addressed whether lentiviral nanoparticles capable of disrupting the EGFP gene and delivering transgene for the CAR expression can be produced. To this end, a lentivector pLenti(sgGFP -CAR[CD19J) containing the template for sgRNA targeting EGFP and the CAR transgene was generated. The lentiviral nanoparticles were transduced to 293GFP cells and the loss of GFP expression followed by UV microscopy and by flow cytometry. It was found that the lentivectors were able to efficiently ablate the EGFP gene, as demonstrated by the loss of EGFP expression in approximately 95% of transduced cells.

Next, a cytotoxicity assay was performed to find out if the VECTR-Cas(sgGFP- CAR[CD19J) lentivector nanoparticles are also able to deliver the CAR transgene to target cells. A T cell lymphoma cell line, SupT1 , was transduced with the VECTR- Cas(sgGFP -CAR[CD19J) and the cells were incubated with lymphocytes expressing a CD19 protein on the cell surface and firefly luciferase (Luc) for precise quantification of living cells. If the transduced SupT1 cells express the CAR[CD19] that recognizes the CD19 protein, lysis of those cells that express the CD19, SupT1 -CD19⁺luc⁺ should be detected. The lysis is manifested as a loss of the Luc expression. The cell lysis should not occur following the co-culture of the SupT1 -CD19⁺luc⁺ cells with mock-transduced SupT 1 cells. The effector (E; SupT 1 -CAR[CD19]) and target (T ; SupT 1 -CD19⁺luc⁺) cells were mixed at various E:T ratios (1 :1 ; 2:1 ; 4:1 ) and the expression of firefly luciferase was followed after 24h to determine the cytotoxicity.

Figure 18 shows cytotoxicity of T cells (SupT1 cell line) transduced with lentivector nanoparticles VECTRv2-Cas(sgGFP)-CAR[CD19] against SupT1 cells that were engineered to express the CD19 and firefly luciferase (SupT1 CD19-luc). The lentivector was used for parallel transductions of HEK293EGFP and SupT1 cells. As shown in Figure 18 (A), the transduction of the HEK293EGFP cells resulted in a loss of the EGFP expression in ~98% of cells. (B) The SupT1 cells transduced with three- component lentivector nanoparticles containing the Cas9 protein, a template for the expression of sgRNA targeting egfp (sgGFP), and the transgene encoding a chimeric antigen receptor specific for CD19 (CAR[CD19]) expressed the CAR[CD19] on the cell surface. This is evidenced by the cytotoxicity of the cells against the cells expressing CD19 antigen (SupT1 CD19-luc). When the transduced cells (effector) were mixed with SupT1 CD19-luc (target) at ratios ranging from 1 :1 to 4:1 , a loss of the firefly luciferase expression (cell killing) was observed. Co-cultivation of the untransduced cells with the target cells did not lead to a reduction of the luciferase expression. SupT 1 CD19-luc cells treated with 1 % Triton X-100 (complete lysis) or media alone (spontaneous lysis) served as experimental controls. Following 24-hour co-culture, cell extracts were made using the cell culture lysis reagent and substrate was added according to instructions for the Luciferase Assay System (Promega). The percentage of killing was calculated as follows: (experimental - spontaneous lysis) x 100 / (complete - spontaneous) lysis.

As shown in Figure 18, co-cultivation of the SupT1-CAR[CD19] cells with SupT1- CD19⁺luc⁺ cells resulted in the killing of the target cells. The greatest cytotoxicity was observed with the highest E:T ratio (4:1). No cytotoxicity was observed for a control co culture of SupT 1 -CD19⁺luc⁺ cells with the untransduced SupT 1 cells.

The data clearly demonstrates that the VECTR-Cas(sgGFP -CAR[CD19J) lentiviral nanoparticles can simultaneously disrupt a gene of interest ( EGFP ) and deliver a transgene (CAR[CD19J) to target cells.

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23 Choi, J. G., Dang, Y., Abraham, S., Ma, H., Zhang, J., Guo, H., Cai, Y., Mikkelsen, J. G., Wu, H., Shankar, P. & Manjunath, N. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther, 23, 627-33 (2016).

24 Cai, Y., Bak, R. O., Krogh, L. B., Staunstrup, N. H., Moldt, B., Corydon, T. J., Schroder, L. D. & Mikkelsen, J. G. DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. Nucleic Acids Res, 42, e28 (2014). 25 Lyu, P., Javidi-Parsijani, P., Atala, A. & Lu, B. Delivering Cas9/sgRNA ribonucleoprotein (RNP) by lentiviral capsid-based bionanoparticles for efficient 'hit-and- run' genome editing. Nucleic Acids Res, 47, e99 (2019).

26 Su K. et al. Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C. Methods, Academic Press, NL, 47(4): 269- 276 (2014).

Claims

1. A lentivirus-based nanoparticle comprising a fusion protein comprising the following structure from N- to C-terminus:

1. a Viral Protein R (Vpr), or a functional derivative thereof,

ii. optionally a protease cleavage site and/or a linker, and

iii. an RNA guided DNA binding polypeptide, preferably an endonuclease, or a functionally active variant thereof.

2. The nanoparticle of claim 1 , comprising the fusion protein and one or more guide RNA (gRNA) templates comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA), or single guide RNA (sgRNA).

3. The nanoparticle of claim 1 or 2, further comprising a transgene, specifically selected from the group consisting of a therapeutic gene, a reporter gene, a gene encoding an enzyme, a gene encoding a pro-drug enzyme, a gene encoding an apoptosis inducer, a gene encoding a fluorescent protein, a gene encoding a pro-drug- activating enzyme, a gene encoding an apoptotic protein, a gene encoding an apoptotic enzyme, a gene encoding a suicide protein, a gene encoding cytokines, a gene encoding an anti-immunosuppressive protein, a gene encoding an epigenetic modulator, a gene encoding a T cell receptor (TCR), a gene encoding a chimeric antigen receptor (CAR), a gene encoding a protein that modifies the cell surface of transduced cells, a gene encoding a protein that modifies the expression of the endogenous TCR, and a gene encoding a switch receptor that converts pro-tumor into anti-tumor signals.

4. The nanoparticle of claim 1 or 2, further comprising a transgene, wherein the transgene comprises a nucleic acid sequenc encoding an RNA sequence capable of RNA interference, specifically pre-miRNA, siRNA or shRNA.

5. The nanoparticle of any one of claims 1 to 4, wherein the RNA guided DNA binding polypeptide is an endonuclease selected from the group consisting of Cas9 and Cpf1.

6. The nanoparticle of any one of claims 1 to 5, wherein the fusion protein further comprises a nuclear localization signal (NLS).

7. The nanoparticle of any one of claims 1 to 6, wherein the nanoparticle comprises

i. a fusion protein, preferably comprising SEQ ID NO:3, comprising the following structure from N- to C-terminus:

a) a Viral Protein R (Vpr),

b) a protease cleavage site,

c) a linker,

d) a nuclear localization signal (NLS), and

e) a Cas 9 endonuclease;

ii. optionally a transgene; and

iii. a gRNA template, which is a sgRNA template.

8. The nanoparticle of any one of claims 1 to 6, wherein the RNA guided binding polypeptide is an endonuclease comprising an enzymatically inactive DNAse domain and is fused to a transcriptional regulator domain, which is a transcriptional activator or a transcriptional repressor, or to an enzyme comprising a deaminase domain.

9. The nanoparticle of any one of claims 1 to 8, wherein Vpr or the functional derivative thereof is derived from a virus belonging to the genus of lentivirus, preferably selected from the group consisting of Human immunodeficiency virus (HIV), specifically HIV-1 or HIV-2, Simian immunodeficiency virus (SIV), Bovine immunodeficiency virus (BIV), Caprine arthritis encephalitis virus, and Maedi-Visna virus.

10. Use of the nanoparticles according to any one of claims 1 to 9 to modify a genomic DNA sequence in a target cell.

11. A method of producing a lentivirus-based nanoparticle, comprising the steps of introducing into a host cell:

i. a first expression construct encoding a transcript comprising:

a) a nucleic acid sequence encoding Viral Protein R (Vpr),

b) optionally a nucleic acid sequence encoding a protease cleavage site and/or a linker between the sequences of a. and c.,

c) a nucleic acid sequence encoding an RNA guided DNA binding polypeptide, preferably an endonuclease, and d) at its 3’ end a stop codon followed by a Rev-response element (RRE); and

11. one or more second expression constructs comprising a gag gene followed by an RRE, a pol gene followed by an RRE, an env gene, and a rev gene; and

maintaining the host cell under conditions allowing formation of the nanoparticle, followed by isolating the nanoparticle.

12. The method of claim 11 , further comprising introducing into the host cell one or more further expression constructs comprising a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA), a transgene, and an RRE.

13. The method of claim 1 1 or 12, wherein the RNA guided DNA binding polypeptide is Cas9 endonuclease.

14. The method of any one of claims 1 1 to 13, comprising the following steps of i. introducing into a host cell the following DNA expression constructs:

a. an endonuclease vector comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a functional derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably Cas9 endonuclease,

b. a gRNA template vector comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA) an RRE, and a transgene, c.an envelope vector comprising a nucleic acid sequence encoding an envelope protein, preferably VSV.G,

d. a packaging vector comprising an RRE and a nucleic acid sequence encoding Gag and Pol polyproteins, optionally including an integrase, and

e. a rev vector comprising a nucleic acid sequence encoding a Rev protein, ii. maintaining the host cell under conditions to allow formation of the nanoparticles, and

iii. isolating the nanoparticles.

15. An expression vector comprising

i. a nucleic acid sequence encoding a fusion protein, comprising from N- to C- terminus: a) a Viral Protein R (Vpr)

b) a protease cleavage site

c) optionally a linker, and/or a nuclear localization signal (NLS), and

d) an RNA guided DNA binding polynucleotide, preferably an endonuclease; and ii. a Rev-response element located 3’ to the nucleic acid sequence encoding the fusion protein.

16. A kit comprising the following plasmids

i. an endonuclease plasmid comprising a Rev-response element (RRE) and a nucleic acid sequence encoding a fusion protein comprising from N- to C-terminus a Vpr, or a derivative thereof, optionally a protease cleavage site, a linker and/or an NLS, and an RNA guided endonuclease, preferably Cas9 endonuclease,

ii. an RNA template plasmid comprising any one or more of CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) or guide RNA (gRNA), optionally a transgene, and an RRE,

iii. one or more plasmids comprising a gag gene, a pol gene, an env gene, a rev gene and, optionally, an RRE.

17. A cell line comprising a polynucleotide encoding the nanoparticle of any one of claims 1 to 9.

18. A cell line comprising the lentiviral-based nanoparticles of any one of claims

1 to 9.

19. A pharmaceutical composition comprising the nanoparticle of any one of claims 1 to 9.