Methods of treating cancer combining UROS invalidation and phototherapy

Abstract

Clinical trials using CRISPR-Cas9 nuclease to ex vivo edit and alter immune cells are ongoing. However, to date, this strategy is not still applied in clinical practice to directly target cancer cells. Here, the inventors propose to mimic a genetic metabolic disorder in cancer cells in order to weaken cancer cells, independently of their genomic abnormalities. Mutations affecting the heme biosynthesis pathway are responsible for porphyria, and most of them are characterized by an accumulation of toxic photoreactive porphyrins. This study aimed to mimic porphyria by using CRISPR-Cas9 to invalidate UROS, leading to porphyrin accumulation in a prostate cancer model. By combining light with gene therapy, the inventors obtained high efficiency in vitro and in vivo, with considerable improvement in the survival of mice. Finally, the inventors achieved the preclinical proof-of-principle of performing cancer CRISPR gene therapy.

Description

[0001] METHODS OF TREATING CANCER COMBINING UROS INVALIDATION AND PHOTOTHERAPY

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular oncology.

[0004] BACKGROUND OF THE INVENTION:

[0005] The evolution of gene therapy is currently generating excitement in many disciplines. Early applications of gene therapy necessarily focused on relatively straightforward genetic disorders, such as severe combined immunodeficiency whose aim was to replace the diseased gene by adding another gene ex vivo using retro / lenti viral vectors. Thereafter, innovative fundamental research and clinical practice led to the development of in vivo AAV-based additive gene therapy for genetic disorders and the launch of innovative cancer gene therapies.1Either alone or in combination with conventional therapies, gene therapy possesses great potential to fight cancer, so cancer management quickly became the main target of gene therapy.2Most protocols involving ex vivo additive gene therapy do not directly target the cancer cells but rather the immune system. T lymphocytes are modified by a chimeric-antigen receptor (CAR) which helps the T cells bind to a specific cancer cell antigen in order to destroy the cancer cells.3Recently, CRISPR-Cas9 technology has revolutionized the gene therapy field. It enables precise insertions or deletions of specific DNA sequences at any point in the target DNA through the initiation of double-strand breaks. In oncology, CRISPR-Cas9 is already used to invalidate PD1 or TCR in CAR-T cells ex vivo in order to boost their anti-tumor activity.4Another way to use CRISPR-Cas9 in cancer gene therapy could be to invalidate a key gene directly in a tumor. However, this promising approach is still underdeveloped.

[0006] In this context, canonical metabolic pathways are promising because they are highly conserved, even within a tumor, and are not dependent on tumor genomic heterogeneity / instability. Mimicking genetic metabolic disorders in cancer cells could be an alternative way to weaken them. Using expertise gained previously,5’6,7,8we propose to mimic porphyria by targeting the heme biosynthesis pathway, which is essential to the proper functioning of the cells and without any possible escape. Heme biosynthesis pathway is a good candidate because i) heme is a key compound in cancer cells9and ii) the blockade of heme biosynthesis results in the accumulation of photo-reactive porphyrins and cell death after light exposure.10Thus, invalidation of the heme biosynthesis pathway by CRISPR-Cas9 could generate an abundant endogenous accumulation of intensely photo-reactive porphyrins in cancer cells. Heme biosynthesis is comprised of eight enzymes whose deficiencies lead to genetic diseases.11In particular, congenital erythropoietic porphyria (CEP) is due to the deficiency of UROS, the fourth enzyme of heme biosynthesis (uroporphyrinogen synthase III, UROS) and induces the most severe skin lesions as a result of exposure to sunlight.12Indeed, it is known that a deficiency of VROS induces a photosensitivity higher than other enzyme deficiencies. Therefore, UROS could be a target of choice to obtain photo-reactive cancer cells. Moreover, unlike other heme enzyme deficiencies that could be directly lethal for cells and too risky, UROS inactivation does not impair cell viability without light exposure, i.e. patients with CEP only have light-induced skin and hematological lesions. Thus, the inactivation of VROS gene by CRISPR-Cas9 does not induce spontaneous cytotoxicity or cell death without irradiation. UROS invalidation could serve as a prodrug in a two-step approach by combining CRISPR editing and tumor illumination. Importantly, gene therapy associated with light exposure offers the opportunity for safe focal treatment with high specificity in illuminated cancer cells.

[0007] Prostate cancer is the second most frequent cancer and the fifth most common cause of cancer mortality in men.13It is a good candidate thanks to its anatomical site which enables direct intra- prostatic injections that facilitate in situ gene therapy protocols and the application of light, ndeed, the first in situ gene therapy clinical trial in prostate cancer started in 1999, and a wide range of gene therapy concepts have been developed since then.14

[0008] SUMMARY OF THE INVENTION:

[0009] The present invention is defined by the claims. In particular, the present invention relates to methods of treating cancer combining UROS invalidation and phototherapy.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] Thanks to its very high genome-editing efficiency, CRISPR-Cas9 technology could be a promising anti -cancer weapon. Clinical trials using CRISPR-Cas9 nuclease to ex vivo edit and alter immune cells are ongoing. However, to date, this strategy is not still applied in clinical practice to directly target cancer cells. Targeting a canonical metabolic pathway essential to the good functioning of the cells without potential escape would represent an attractive strategy. Here, the inventors propose to mimic a genetic metabolic disorder in cancer cells in order to weaken cancer cells, independently of their genomic abnormalities. Mutations affecting the heme biosynthesis pathway are responsible for porphyria, and most of them are characterized by an accumulation of toxic photoreactive porphyrins. This study aimed to mimic porphyria by using CRISPR-Cas9 to invalidate UROS, leading to porphyrin accumulation in a prostate cancer model. Prostate cancer is currently the leading cancer in men and has a high mortality rate despite therapeutic progress, with a primary tumor accessible to light. By combining light with gene therapy, the inventors obtained high efficiency in vitro and in vivo, with considerable improvement in the survival of mice. Finally, the inventors achieved the preclinical proof-of- principle of performing cancer CRISPR gene therapy.

[0012] Main definitions:

[0013] As used herein, the term “UROS” has its general meaning in the art and refers to the uroporphyrinogen synthase III encoded by the UROS gene (Gene ID:7390). An exemplary amino acid sequence for UROS is shown as SEQ ID NO:1.

[0014] SEQ ID NO : 1 >sp | Pl 0746 | HEM4 HUMAN Uroporphyrinogen-I I I synthase 0S=Homo sapiens OX=9606 GN=UROS PE=1 SV=1 MKVLLLKDAKEDDCGQDPYIRELGLYGLEATLI PVLSFEFLSLPSFSEKLSHPEDYGGLI FTSPRAVEAAELCLEQNNKTEVWERSLKEKWNAKSVYWGNATASLVSKIGLDTEGETCG NAEKLAEYICSRESSALPLLFPCGNLKREILPKALKDKGIAMESITVYQTVAHPGIQGNL NSYYSQQGVPASITFFSPSGLTYSLKHIQELSGDNIDQIKFAAIGPTTARALAAQGLPVS CTAESPTPQALATGIRKALQPHGCC

[0015] As used herein, the term “porphyrin” has its general meaning in the art and refers to a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their a carbon atoms via methine bridges (=CH-). In particular, the term “photoreactive porphyrin” refers to porphyrins having the capacity on exposing red fluorescence when exposed to long- wavelength light that ranges from 400nm to 700nm as described in the EXAMPLE.

[0016] As used herein the term "increasing the content of photoreactive porphyrins" in a cancer cell indicates that the content photoreactive porphyrin is at least 5% higher in the cancer cell treated with the DNA-targeting endonuclease, than in a comparable, cancer cell, wherein an endonuclease targeting an unrelated locus is present or where no endonuclease is present. In some embodiments, the content of photoreactive porphyrins in the cancer cell is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10 fold higher, at least 100 fold higher, at least 1000-fold higher, or more than a cancer cell, wherein an endonuclease targeting an unrelated locus is present or where no endonuclease is present. In some embodiments, any method known in the art can be used to measure an increase in photoreactive porphyrins and typically those described in the EXAMPLE.

[0017] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product”. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Any method known in the art can be used to measure the expression of the gene (e. g. WESTERN analysis of protein and RT-qPCR analysis of mRNA).

[0018] As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. The term "substitution" means that a specific nucleotide at a specific position is removed and another nucleotide is inserted into the same position. The term "deletion" means that a specific nucleotide is removed. The term "insertion" means that one or more nucleotides are inserted before or after a specific nucleotide.

[0019] As used herein, the term “editing” in the context of editing of a genome of a cell refers to inducing a mutation in the sequence of the genome at a target genomic region. For example, the editing can take the form of inducing an insertion deletion (indel) mutation into a sequence of the genome at a target genomic region. Such editing can be performed by inducing a double stranded break within a target genomic region, or a pair of single stranded nicks on opposite strands and flanking the target genomic region. The term “editing” also includes cleavage of the target gene; this may be required for downstream editing applications, e.g. NHEJ, Homology directed repair with donor template, etc. As used herein, the term “prime editing” refers to the “search-and-replace” genome editing technology that was first disclosed in Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA. "Nature 576. 7785 (2019): 149-157. The technology directly writes new genetic information into a targeted DNA site. The technology can mediate targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. As disclosed herein, the “gene editing platform” comprises all the elements for performing said editing and typically include one or more base-editing enzymes or one or more prime-editing enzymes and one or more guide RNAs.

[0020] As used herein, the term "DNA targeting endonuclease" has its general meaning in the art and refers to an endonuclease that generates a double-strand break (DSB) at a desired position in the genome without producing undesired toxic off-target DSBs. The DNA targeting endonuclease can be a naturally occurring endonuclease (e.g., a bacterial meganuclease) or it can be artificially generated (e.g., engineered meganucleases, TALENs, or ZFNs, CRISPR- associated endonuclease, among others).

[0021] As used herein the term "cleaves" generally refers to the generation of a double- strand break in the DNA genome at a desired location. The term “cleavage site” refers to any site in a target sequence that can be cleaved by a DNA targeting endonuclease. Cleavage thus results in alteration of the genome sequence by non-homologous end joining (NHEJ) repair system or microhomology mediated end joining (MMEJ) repair system.

[0022] As used herein, the term “TALEN” has its general meaning in the art and refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit a target gene. TALENs are produced artificially by fusing a TAL effector (“TALE”) DNA binding domain, e.g., one or more TALEs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TALEs to a DNA- modifying domain, e.g., a FokI nuclease domain. Transcription activator-like effects (TALEs) can be engineered to bind any desired DNA sequence (Zhang (2011), Nature Biotech. 29: 149- 153). By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence. These can then be introduced into a cell, wherein they can be used for genome editing (Boch (2011) Nature Biotech. 29: 135- 6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501). TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence (Zhang (2011), Nature Biotech. 29: 149-153). To produce a TALEN, a TALE protein is fused to a nuclease (N), e.g., a wild-type or mutated FokI endonuclease. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity (Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786- 793; and Guo et al. (2010) J. Mol. Biol. 200: 96). The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity (Miller et al. (2011) Nature Biotech. 29: 143-8). TALEN can be used inside a cell to produce a double-strand break in a target nucleic acid, e.g., a site within a gene. A mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non- homologous end joining (Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16). For example, improper repair may introduce a frame shift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene via the homologous direct repair pathway, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene.

[0023] As used herein, the term “ZFN” or “Zinc Finger Nuclease” has its general meaning in the art and refers to a zinc finger nuclease, an artificial nuclease which can be used to edit a target gene. Like a TALEN, a ZFN comprises a DNA-modifying domain, e.g., a nuclease domain, e.g., a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 zinc fingers (Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160). A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art (Sera (2002), Biochemistry, 41 :7074-7081; Liu (2008) Bioinformatics, 24: 1850-1857). AZFN using aFokl nuclease domain or other dimeric nuclease domain functions as a dimer. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5). Also like a TALEN, a ZFN can create a DSB in the DNA, which can create a frame-shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell.

[0024] As used herein, the term “CRISPR-associated endonuclease” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In bacteria the CRISPR / Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types (I- VI) of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR-associated endonucleases Cas9 and Cpfl belong to the type II and type V CRISPR / Cas system and have strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 nucleotides of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rdor the 4thnucleotide from PAM). The crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion small guide RNA (sgRNA) via a synthetic stem loop to mimic the natural crRNA / tracrRNA duplex. Such sgRNA, like shRNA, can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6 or Hl -promoted RNA expression vector.

[0025] In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. The Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus,' Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms. Alternatively, the wild type Streptococcus pyogenes Cas9 sequence can be modified. The nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., "humanized." A humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765. Alternatively, the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as pX330, pX260 or pMJ920 from Addgene (Cambridge, MA). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of pX330, pX260 or pMJ920 (Addgene, Cambridge, MA).

[0026] As used herein, the term “guide RNA” or “gRNA” has its general meaning in the art and refers to an RNA which can be specific for a target DNA and can form a complex with the CRISPR- associated endonuclease. A guide RNA can comprise a spacer sequence that specifies a target site and guides an RNA / Cas complex to a specified target DNA for cleavage. Site-specific cleavage of a target DNA occurs at locations determined by both 1) base-pairing complementarity between a guide RNA and a target DNA (also called a protospacer) and 2) a short motif in a target DNA referred to as a protospacer adjacent motif (PAM). The sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S. pyogenes, the target DNA typically immediately precedes a 5'-NGG proto-spacer adjacent motif (PAM). Thus, for the S. pyogenes Cas9, the PAM sequence can be AGG, TGG, CGG or GGG. Other Cas9 orthologs may have different PAM specificities. The specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency of alteration at the targeted loci. The length of the spacer sequence can vary from about 17 to about 60 or more nucleotides, for example about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides. The guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs. As used herein, the term “base-editing enzyme” refers to fusion protein comprising a defective CRISPR / Cas nuclease linked to a deaminase polypeptide. Two classes of base-editing enzymes — "cytosine base-editing enzymes” (CBEs) and “adenine base-editing enzymes” (ABEs)— can be used to generate single base pair edits without double stranded breaks. Typically, base-editing enzyme are created by fusing the defective CRISPR / Cas nuclease to a deaminase.

[0027] As used herein, the term “deaminase” refers to an enzyme that catalyses a deamination reaction. The term “deamination”, as used herein, refers to the removal of an amine group from one molecule. In some embodiments, the deaminase is a “cytidine deaminase”, catalysing the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase is an “adenosine deaminase”, catalysing the hydrolytic deamination of adenosine to inosine, which is treated like guanosine by the cell, creating an A to G (or T to C) change.

[0028] As used herein, the term “prime editing enzyme” refers to a fusion protein comprising a defective CRISPR / Cas nuclease linked to a reverse transcriptase. The term is also known as “prime editor”.

[0029] As used herein, the terms “prime editing guide RNA” or “pegRNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing as described herein. Typically, the pegRNAs of the present invention comprise in the 5’ to 3’ direction a spacer, a gRNA core, and an extension arm. In some embodiments, the pegRNA of the present invention may also further comprise elements, such as, but not limited to aptamers, stem loops, hairpins, toe loops (e.g., a 3’ toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin). In particular, the pegRNA may contain one or more structural elements for minimizing its degradation. In particular, the pegRNA of the present invention incorporates one or more stable pseudoknots at its 3’ end such as a modified prequeosinel-1 riboswitch aptamer (evopreQl) or the frameshifting pseudoknot from Moloney murine leukemia virus (MMLV), hereafter referred to as “mpknof ’ as described in Nelson, James W., et al. "Engineered pegRNAs improve prime editing efficiency. " Nature biotechnology 40.3 (2022): 402-410 for which the teaching is incorporated by reference. In some embodiments, the pegRNA may comprise a transcriptional termination sequence at the 3’ of the molecule. As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0030] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0031] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0032] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0033] As used herein, the term “Therapeutically effective amount” refers to the level or amount of the DNA-targeting endonuclease thereof as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.

[0034] As used herein, the term “about” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%.

[0035] Methods of the invention:

[0036] The first object of the present invention relates to a method of treating cancer in a patient in need thereof comprising the steps of i) of increasing the content of photo-reactive porphyrins in cancer cells by invalidating the expression of UROS gene in said cancer cells and ii) lightactivating the photo-reactive porphyrins in cancer cells to produce cytotoxic species, thereby inducing the death of cancer cells.

[0037] In some embodiments, the method of the present invention comprises the steps of i) of increasing the content of photo-reactive porphyrins in cancer cells by editing the cancer cells to invalidate the expression of UROS gene in said cancer cells and ii) light-activating the photo- reactive porphyrins in cancer cells to produce cytotoxic species, thereby inducing the death of cancer cells.

[0038] The editing can be performed by any method well known in the art, and typically includes the techniques herein disclosed. i) Use of DNA-targeting endonucleases

[0039] In some embodiments, the editing can involve the use of a DNA-targeting endonuclease that cleaves the genomic DNA of the cancer cells in at least one position located in the targeted gene (i.e. UROS). This system leads to a DNA double-strand break (DSB) that subsequently leads to non-conservative non-homologous end-joining (NHEJ) repair pathway. Insertions or deletions (indels) at the on-target site thus cause frameshifts in open reading frames and repress the expression of the targeted gene. Thus, in some embodiments, the present invention comprises the steps of administering to the patient a therapeutically effective amount of a DNA-targeting endonuclease that cleaves the genomic DNA of the cancer cells in at least one position located in UROS gene thereby invalidating the expression of said gene in said cancer cells.

[0040] In some embodiments, the DNA targeting endonuclease of the present invention is a TALEN.

[0041] In some embodiments, the DNA targeting endonuclease of the present invention is a ZFN.

[0042] In some embodiments, the DNA targeting endonuclease of the present invention is a CRISPR- associated endonuclease.

[0043] In some embodiments, nucleotide sequence encoding for the nuclease (e.g. Cas9) can be modified to encode biologically active variants of said nuclease, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type nuclease by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations). One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a nuclease polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type nuclease polypeptide. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. The nuclease sequence can be a mutated sequence. For example the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage. For example, an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks.

[0044] In some embodiments, the method of the present invention comprises the step of contacting the cancer cell with an effective amount of a CRISPR-associated endonuclease and with one or more guide RNA. In some embodiments, the guide RNA is used for recruiting the CRISPR-associated endonuclease to the UROS gene and generating a DSB in exon 4. In some embodiments, the guide RNA is used for recruiting the CRISPR-associated endonuclease to the UROS gene and generating a DSB between nucleotides at position 49 and 50. In some embodiments, the guide RNA comprises a spacer sequence capable of annealing to the sequence as set forth in SEQ ID NO:2 (i.e. GCAGCAGAGTTATGTT). In some embodiments, the guide RNA comprises the spacer sequence as set forth in SEQ ID NO:3 (i.e. GGAAGCAGCAGAGTTATGTT) for recruiting the CRISPR-associated endonuclease to the UROS gene and generating doublestrand breaks between positions 49 and 50. ii) Use of base-editing enzymes and prime-editing enzymes

[0045] In some embodiments, the editing can involve the use of a gene-editing platform to introduce a stop codon into the coding sequence of targeted gene (i.e. UROS) upstream of the normal stop codon (referred to as a “premature stop codon”). Premature stop codons thus cause premature translation termination, in turn resulting in truncated and nonfunctional proteins and induces rapid degradation of the mRNA via the non-sense mediated mRNA decay pathway. See, e.g., Baker et al., Current Opinion in Cell Biology 16 (3): 293-299, 2004; Chang et al., Annual Review of Biochemistry 76: 51-74, 2007; and Behm-Ansmant et al., Genes & Development 20 (4): 391-398, 2006, each of which is incorporated herein by reference.

[0046] Thus, in some embodiments, the method of the present invention comprises the steps of contacting the cancer cells with a gene-editing platform that consists of a (a) at least one baseediting enzyme and (b) least one guide RNA molecule for guiding the base-editing enzyme to at least one target sequence in the UROS gene and thereby repressing the expression of said gene.

[0047] In some embodiments, the base-editing enzyme of the present invention comprises a defective CRISPR / Cas nuclease. The sequence recognition mechanism is the same as for the nondefective CRISPR / Cas nuclease. Typically, the defective CRISPR / Cas nuclease of the invention comprises at least one RNA binding domain. The RNA binding domain interacts with a guide RNA molecule as defined hereinafter. However, the defective CRISPR / Cas nuclease of the invention is a modified version with no nuclease activity. Accordingly, the defective CRISPR / Cas nuclease specifically recognizes the guide RNA molecule and thus guides the base-editing enzyme to its target DNA sequence. In some embodiments, the CRISPR / Cas nuclease consists of a mutant CRISPR / Cas nuclease i.e. a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In some embodiments, the mutant has the RNA-guided DNA binding activity, but lacks one or both of its nuclease active sites. In some embodiments, the CRISPR / Cas nuclease is a mutant of a wild type CRISPR / Cas nuclease (such as Cas9) or a fragment thereof. In some embodiments, the CRISPR / Cas nuclease is a mutant Cas9 protein from S. pyogenes. Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA- Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). In some embodiments, the CRISPR / Cas nuclease of the present invention is nickase and more particularly a Cas9 nickase i.e. the Cas9 from S. pyogenes having one mutation selected from the group consisting of D10A and H840A.

[0048] According to the present invention, the second component of the base-editing enzyme herein disclosed comprises a non-nuclease DNA modifying enzyme that is a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC 1 family deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the deaminase is an ACF1 / ASE deaminase. In some embodiments, the deaminase is selected from the group consisting of AID: activation induced cytidine deaminase, APOBEC 1 : apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 1, APOBEC3A: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 A, APOBEC3B: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B, APOBEC3C: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3C, APOBEC3D: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3D, APOBEC3F: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F, APOBEC3G: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, APOBEC3H: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3H, ADA: adenosine deaminase, AD ARI : adenosine deaminase acting on

[0049] RNA 1, Dnmtl : DNA (cytosine-5-)-m ethyltransferase 1, Dnmt3a: DNA (cytosine-5-)- methyltransferase 3 alpha, Dnmt3b: DNA (cytosine-5-)-m ethyltransferase 3 beta and Tetl : methylcytosine dioxygenase. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an ADAT family deaminase. In some embodiments, the adenosine deaminase variant is a TadA deaminase. In some embodiments, the adenosine deaminase variant is a Staphylococcus aureus TadA, a Bacillus subtilis TadA, a Salmonella typhimurium TadA, a Shewanella putrefaciens TadA, a Haemophilus influenzae F3031 TadA, a Caulobacter crescentus TadA, or a Geobacter sulfurreducens TadA, or a fragment thereof. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase.

[0050] In some embodiments, the deaminase is fused to the N-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the deaminase is fused to the C-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the defective CRISPR / Cas nuclease and the deaminase are fused via a linker.

[0051] Various base-editing enzymes are known in the art (see e.g. Improving cytidine and adenine base-editing enzymes by expression optimization and ancestral reconstruction. Nat Biotechnol. 2018 May 29) and typically include AB Emax, AncBE4max, evoCDAl-BE4max-NG, evoFERNY-BE4max, CBE-NRCH, CBE-SpG, ABE-SpRY, CBE-SpRY, ABE8e, ABE8e- SpRY, ABE8e-NRCH and NG-ABE8e.

[0052] The second component of the gene-editing platform disclosed herein consists of at least one guide RNA molecule suitable for guiding the base-editing enzyme to at least one target sequence located in the UROS gene.

[0053] In some embodiments, the method of the present invention comprises the step of contacting the cancer cell with a gene-editing platform that consists of (a) one prime editing enzyme and (b) one prime editing guide RNA (pegRNA) for guiding the prime editing enzyme to one target nucleic acid sequence in the UROS gene, thereby prime editing said region and subsequently repressing the expression of UROS in said cancer cells.

[0054] In some embodiments, the prime editing enzyme of the present invention comprises a defective CRISPR / Cas nuclease as described above for the base-editing enzyme.

[0055] According to the present invention, the second component of the prime editing enzyme herein disclosed comprises a reverse transcriptase. The disclosure contemplates any wild type reverse transcriptase obtained from any naturally-occurring organism or virus, or obtained from a commercial or non-commercial source. In addition, the reverse transcriptases can include any naturally-occurring mutant RT, engineered mutant RT, or other variant RT, including truncated variants that retain function. The RTs may also be engineered to contain specific amino acid substitutions, such as those specifically disclosed herein. A person of ordinary skill in the art will recognize that wild type reverse transcriptases, including but not limited to, Moloney Murine Leukemia Virus (M-MLV); Human Immunodeficiency Virus (HIV) reverse transcriptase and avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase may be suitably used in the subject methods and composition described herein.

[0056] In some embodiments, the reverse transcriptase is fused to the N-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the reverse transcriptase is fused to the C- terminus of the defective CRISPR / Cas nuclease.

[0057] Various prime editing enzymes are known in the art and typically include the PEI, PE2, PEmax, PEmax-SpRY prime editors.

[0058] The second component of the prime editing platform disclosed herein consists of a pegRNA suitable for guiding the prime editing enzyme to one target sequence located in the UROS gene.

[0059] According to the present invention, the pegRNA comprises (a) a spacer sequence that comprises a region of complementarity to a first strand of the double-stranded target nucleic sequence located in UROS gene; (b) an extension arm that comprises a RT template and a primer binding site in a 5’ to 3’ orientation, wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in the second strand of the double-stranded target sequence, and wherein the RT template encodes the desired nucleotide changes compared to a region downstream of the nick site in the second strand of the double-stranded target sequence.

[0060] In some embodiments, the prime editing platform further involves the use of a second strand nicking guide RNA (“nicking guide RNA” or ngRNA”) that complexes with the prime editing platform and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand (PE3 system). In particular, the ngRNA is designed for temporal control. As used herein, the term “temporal second-strand nicking” refers to a variant of second strand nicking whereby the installation of the second nick in the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands that could lead to double-stranded DNA breaks. This is achieved by designing a ngRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the ngRNA until after the editing event on the PAM strand takes place.

[0061] Typically, the components for performing the editing of the cancer cells (e.g. DNA-targeting endonucleases, base-editing enzymes, prime-editing enzymes, gRNAs. . .) are provided through expression from one or more expression vectors.

[0062] For instance, the CRISPR endonuclease can be encoded by the same nucleic acid as the guide RNA sequences. Alternatively or in addition, the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the guide RNA sequences or in a separate vector.

[0063] Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA. In some embodiments, the vector is a viral vector such as an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and / or env genes but without the LTR and / or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector of the present invention include “control sequences”, which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'- direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. In some embodiments, the promoter is a prostatic cancer promoter such as DD3 / PCA3 as described in the EXAMPLE.

[0064] In some embodiments, the components for performing the editing (e.g. DNA-targeting endonucleases, base-editing enzymes, prime-editing enzymes, gRNAs...) are provided to the population of cells through the use of an RNA-encoded system. For instance, the editing system may be provided to the population of cells through the use of a chemically modified mRNA. In particular said modifications consist in uridine depleted mRNAs modified with 5- methoxyuridine: synonymous codons may be introduced to deplete uridines as much as possible without altering the coding sequence and replaced all the remaining uridines with 5- methoxyuridine. Said optimized editing system exhibits higher editing efficiency at some genomic sites compared to DNA-encoded system. It is also possible to encapsulate the modified mRNA and guide RNA into lipid nanoparticle (LNP) for allowing lipid nanoparticle (LNP)- mediated delivery. In some embodiments, the components for performing the editing (e.g. DNA-targeting endonucleases, base-editing enzymes, prime-editing enzymes, gRNAs...) are provided to the population of cells through the use of ribonucleoprotein (RNP) complexes. For instance the DNA-targeting endonucleasecan be pre-complexed with one or more gRNAs to form a ribonucleoprotein (RNP) complex. The RNP complex can thus be introduced into the cancer cells. Introduction of the RNP complex can be timed. The cell can be synchronized with other cells at Gl, S, and / or M phases of the cell cycle. RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery. Typically, the RNP complex is produced simply by mixing the proteins (e.g. DNA-targeting endonuclease) and one or more gRNAs in an appropriate buffer. This mixture is incubated for 5-10 min at room temperature before electroporation. Electroporation is a delivery technique in which an electrical field is applied to one or more cells in order to increase the permeability of the cell membrane. In some embodiments, genome editing efficiency can be improved by adding a transfection enhancer oligonucleotide.

[0065] In some embodiments, it may be desirable to administer the components for performing the editing (e.g. DNA-targeting endonucleases, base-editing enzymes, prime-editing enzymes, gRNAs. . .) locally to the area in need of treatment; this can be achieved, for example, by local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.

[0066] Typically, the photo-reactive porphyrins can be activated at the target site with lasers or other light sources via optical fibres or any other appropriate method. For instance, the photoactivating light can be delivered to the target site from a conventional light source or from a laser. Target tissues are illuminated, usually with red light from a laser. Typically, the wave length ranges from 400nm to 700nm. However, given that red and / or near infrared light best penetrates mammalian tissues, a wavelength of about 530nm may preferred as described in the EXAMPLE. Typically, the dose of light ranges from 10J / cm2to 50J / cm2, preferably 20J / cm2. Delivery can be direct, by transillumination, or by optical fiber. Optical fibers can be connected to flexible devices such as balloons equipped with light scattering medium. Flexible devices can include, for example, laparoscopes, arthroscopes and endoscopes. Thus in some embodiments, the photodynamic therapy of the present invention is performed by photodynamic therapy by coelioscopy or laparoscopy.

[0067] The method of the present invention is in particularly useful for the treatment of patients suffering from prostate cancer.

[0068] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0069] FIGURES:

[0070] Figure 1. UROS inactivation combined with repeated 530nm light induces PC3 tumor regression in vivo. (A) Experimental design. Luc+PC3 (blue) and UROS KO Luc+PC3 (red) cells were subcutaneously injected in NSG mice (one side), and irradiated (or not) three times per week (Monday, DO-Wednesday, D2-Friday, D4) for one week at 530nm. Illumination is indicated by the purple light. Six mice per group. Follow-up of BLI, tumor volume and mice survival for 17 days (sacrifice). (B) Monitoring of tumor growth by BLI quantification over time (left) and at day 14 (right). Mean + / - SEM. Statistical differences were determined by Mann-Whitney post Kruskal-Wallis test. NS, not significant, **, p<0.01. (C) Volume of tumor growth over time (left) or at day 14 (right). Crosses mean that all mice in a group are dead. Mean+ / - SEM. Statistical differences were determined by Mann-Whitney post Kruskal-Wallis test. NS, not significant, *, p<0.05, **, p<0.01. (D) Absence of dermotoxicity with repeated 530nm illumination (E) Kaplan-Meier plot for survival of mice with PC3 tumors treated or not by LV-UROS-Cas9 and repeated 530nm light. Statistical difference by Mantel-Cox test.

[0071] Figure 2. UROS inactivation in PC3 cells combined with long-term repeated 530nm light increases mice survival. (A) Experimental design. Luc+PC3 (blue) and UROS KO Luc+PC3 (red) cells were subcutaneously injected in mice (one side) and irradiated for URO O three times per week for five weeks at 530nm. Illumination is indicated by purple light. Five mice per group. (B) Volume of tumor over time. Crosses mean that all mice in a group are dead. Mean+ / - SEM. (C) Kaplan-Meier plot for survival of mice with PC3 tumors treated or not by LV-UROS-Cas9 and repeated long-term 530nm light. Statistical difference by Mantel-Cox test. EXAMPLE:

[0072] Material & Methods:

[0073] Cell culture

[0074] PC3 cell line was a gift from Mariangela Figini lab.47This cell line is modified and stably expresses PSMA (PC3-PIP), as developed by W. Heston (Cleveland, OH).48Cells were maintained in vitro at 37°C in a humidified atmosphere of 5% CO2 / 95% air in RPMI 1640, glutamax (Gibco® by ThermoFisher Scientific, Carlsabad, CA, USA), 10% fetal bovine serum (FBS), and 1% Pen-Strep (Eurobio Scientific, Les Ulys, France). Cell lines were maintained Mycoplasma-R through monthly testing by PCR. For in vivo bioluminescence monitoring, cells (PSMA+PC3 PIP (PC3) and UROS KO PSMA+ PC3 PIP (UROS KO PC3)) were stably transfected using lipofectamine (Invitrogen) with a plasmid coding for luciferase under the control of CMV promoter (pcDNA6.2-CMV),49and selected by blasticidin (lOpg / mL, Euromedex, Souffelweyersheim, France).

[0075] Lentivector construction and production

[0076] Lentivirus vector was produced by the Vect'UB service platform, (INSERM US 005 - CNRS UMS 3427- TBM-Core, Universite de Bordeaux, France). We designed a lentiviral vector (named LV-UROS-Cas9) containing a gRNA against exon 4 UROS (GGAAGCAGCAGAGTTATGTT) under the control of the U6 promoter, CRISPR-Cas9 nuclease DNA under the control of the EFl alpha promoter, and the ZsGreen reporter to control transduction efficiency. LentiCRISPR v2 (Z$hang lab) was modified to replace the PuroR gene with the ZsGreen gene. LentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid # 52961 ; http: / / n2t.nct / addgene:52961 ; RRID:Addgene_52961). Synthetic gRNA oligonucleotides were cloned into pLentiCRISPR-ZsGreen modified vector at BsmBI restriction sites.

[0077] VROS gene editing in PC3 cells and tumors

[0078] For the in vitro study, we transduced PC3 cells with LV-UROS-Cas9 particles (multiplicity of infection (MOI) 20). We monitored cell transduction efficiency by cytometry using Zs-Green reporter expression. To validate CRISPR double-strand break efficiency in PC3 cells and the presence of insertion / deletions (Indels) in the UROS gene, genomic DNA of transduced PC3 cells was extracted using Nucleospin® Tissue (Macherey-Nagel®) according to the manufacturer’s protocol. The genomic region flanking the expected cut-site was amplified by PCR (HotStarTaq Plus DNA polymerase, Qiagen®, Venlo, Netherlands) with human primers UROS F TAGTTCCAGGCACATAGTAAGCAC, and UROS R AGGAGGTGAACAACGAATAGACAG. PCR products were purified with Nucleospin® Gel and PCR Clean-up (Macherey -Nagel). To check the presence of UROS KO cells in tumors with or without illumination, genomic DNA of tumors was extracted with the Maxwell RSC DNA FFPE Kit (Promega, Charbonnieres-les-Bains, France). Extracted DNA was eluted in 70 pl of nuclease-free water and DNA concentration was determined by fluorimetry with the DS11FX automated system (DeNovix). The genomic region flanking the expected cut-site was amplified by PCR (HotStarTaq Plus DNA polymerase, Qiagen®, Venlo, Netherlands) with human primers UROS F GGTGTGCAGCTTTCTCATCC, and UROS R AAACTGAAGGTGAGGGTGGG. PCR products were purified with Nucleospin® Gel and PCR Clean-up (Macherey-Nagel). Sanger sequencing was done on purified PCR products and sequenced by LIGHTRUN (GATC Biotech, Konstanz, Germany). Sanger sequencing data were analyzed using ICE v2 CRISPR Analysis tool (ICE) software (Synthego, Redwood City, USA). Purified PCR products from non-edited cells were used as control chromatogram.

[0079] Porphyrin accumulation in PC3 cells

[0080] Porphyrins are red-fluorescent compounds. Intracellular total porphyrin contents were determined after extraction with methanol / perchloric acid 1 M (30 / 70 vol / vol) and quantified by spectrofluorometry (Hitachi F-4500 fluorescence spectrophotometer) using commercially available calibrators (ClinCal Urine Calibrator Recipe). Cells were also analyzed at 550 nm emission wavelength with a 405 nm excitation laser on a FACS flow cytometer (Per-CP chanel, BD Biociences Accuri C6 Plus apparatus) and the data were analyzed with BD CSamplerTM software (BD Biosciences, Le Pont de Claix, France).

[0081] In vitro illumination of PC3 cells

[0082] PSMA+PC3 PIP cells were plated at 105cells per well in a 48-well plate and incubated overnight. The next day, cells were either irradiated, without cover, by a system with interchangeable LED light sources (Thorlabs, wavelengths 405nm or 530nm or 660nm) or were not irradiated. To control the light source, we monitored light fluence by a lightmeter (Thorlabs) and we protected the other wells from light with an opaque mask. Cell viability was measured by cell count 72 h post-irradiation. Tumor generation and in vivo illumination

[0083] All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Bordeaux University (agreement #25312) and complied with the French and European regulations on Animal Welfare and Public Health Service. Immunodeficient male NSG (NOD / SCID / IL2Rynull) mice were housed at the Bordeaux University facility and maintained under 12hdark / light cycles with water and food provided and libitum. Human PC3 cells (2.10* 100 pL) were implanted subcutaneously in 10-week-old NSG mice on the top of the back (flanks). One week after cell injection when the tumor diameter had reached 3-5mm, mice were shaved with clippers and treated with illumination with the same system as for in vitro illumination. For irradiation, the mouse was placed on a thermostatically controlled bed, and its skin was protected from light by an opaque mask that had a 10mm hole to allow the tumor to be irradiated. The light source was positioned 4cm from the skin and irradiation was performed for 20 minutes.

[0084] Antitumor Efficacy in Subcutaneous Xenograft Model

[0085] Tumor progression was monitored either by measuring subcutaneous tumor volume using the formula [volume = 7t / 6 .f. (length. width)3 / 2)mm3, f= 1.69 for male],50using a caliper, or by in vivo bioluminescence imaging at the Vivoptic platform (Univ. Bordeaux, CNRS, INSERM, TBM-Core, UAR 3427, US5, F33000 BORDEAUX, France). Bioluminescence imaging was performed using the Lumina LT Imaging system (Perkin Elmer Inc., Boston, MA, USA). D- luciferin (Promega, 2.9mg / 100pL PBS) was injected intraperitoneally and bioluminescence acquisition (1 min 4x4 binning) and photographs (100ms) were taken 8 minutes after substrate injection. Data were analyzed with Living Image software (Perkin Elmer). After sacrifice, tumor weight was evaluated using a Sartorius balance. Mice were euthanized and scored as death if tumor diameter was > 10mm or if mice lost more than 10% of body weight or had signs of discomfort, such as hunched posture, anorexia, or dehydration. For both animal models, the probability of survival was plotted by Kaplan-Meier curves using Prism software (GraphPad Software). Tumors were fixed in 10% neutral buffered formalin and embedded in paraffin.

[0086] Statistical analysis

[0087] Statistical significance was inferred when necessary. Exact distinct and independent sample size is indicated in each legend (n). Graph Pad Prism 6 software was used for statistical analysis. Results are presented as mean ± SD for in vitro experiments and mean ± SEM for in vivo experiments. The parametric T-test was used when distribution was Gaussian / normal (Shapiro- Wilk test). The non-parametric Mann-Whitney test (two-sided) was used to compare two groups. One-way ANOVA, complemented with the unprotected Fisher’s Least Significant Difference test, was used to compare more than two groups. Survival analyses were performed using Prism software (version 9; GraphPad Software). P values were calculated by the log-rank (Mantel-Cox) test and were considered significant if less than 0.05.

[0088] Results:

[0089] CRISPR-Cas9-mediated VROS inactivation sensitizes prostate cancer PC3 cells to 405nm-light in vitro

[0090] The first step was to inactivate UROS in order to induce photoreactive red-fluorescent type-I porphyrin accumulation in cells (data not shown). We designed a lentiviral vector containing the CRISPR-Cas9 nuclease sequence, a gRNA against UROS and a Zs-Green reporter sequence to visualize transduction efficiency by flow cytometry (data not shown). We efficiently transduced the human prostatic cancer cell line (PC3) expressing PSMA (PC3-PIP), with up to 95% of cells expressing Zs-Green detected by cytometry (UROS KO PC3 cells, data not shown). Transduction of this vector induced a high rate of Indels (>90%) in the targeted UROS (data not shown). To evaluate whether UROS inactivation produces porphyrin accumulation (fluorescent in Per-CP channel), we monitored the kinetic of the appearance of fluorescent PerCP cells in UROS KO PC3 cells. Fourteen days after UROS gene editing, we achieved >90% Per-CP+cells by cytometry (data not shown) and confirmed the high porphyrin accumulation in cells by spectrofluorimetry (data not shown). Porphyrin detection was stable over time, i.e. at least 26 days. These data validated the CRISPR approach to edit UROS and to induce the endogenous accumulation of porphyrins. To test the photosensitivity of PC3 cells after UROS editing, we performed illumination at 405nm, their major type-I porphyrin absorbance wavelength (Soret band). We carried out a scale-dose of light from 2J to 20J / cm2in WT and UROSKO PC3 cells to evaluate the cell death induced. From 2J / cm2(corresponding to 1 minute of light), light induced the cell death of UROS KO PC3 cells (higher than 95%, p<0.001 compared to WT cells with light). Importantly, all UROS KO PC3 cells died with 20J / cm2, without any toxicity in WT PC3 cells (data not shown). Altogether, these data demonstrate the efficiency of UROS inactivation coupled with a 405 nm light exposure to induce the death of prostate cancer cells. UROS inactivation sensitizes prostate cancer PC3 cells to 405nm-light in vivo but induces dermotoxicity

[0091] We next decided to confirm our in vitro results in an in vivo 3D model. To monitor tumor growth, we modified human PC3 cells (WT and UROS KO) to express luciferase to obtain a non-invasive and sensitive bioluminescence-based tumor quantification method. We subcutaneously xenografted PC3 cells on the two contralateral flanks of immune-deficientNSG mice to obtain either two tumors with WT PC3 cells or two tumors with UROS KO PC3 cells per mouse. After seven days, we illuminated only one tumor of each mouse with a single dose of 405nm-light (20J / cm2, corresponding to 8 minutes). To measure illumination efficacy, we measured the bioluminescence of tumors for four days after treatment and weighed the tumors at sacrifice at day 4 (data not shown). Whereas illumination did not modify the tumor growth in WT PC3 tumors, 405nm-light drastically reduced bioluminescence intensity in UROS KO PC3 tumors (data not shown). The weight of tumors at sacrifice four days after illumination confirmed the specific effect of 405nm-light on UROS KO PC3 tumors (data not shown). Indeed, with only one dose of 405nm -light, UROS O PC3 tumors were three-fold smaller than non-illuminated UROS O PC3 control ones. No significant effect of lighting was observed in WT PC3 tumors. These in vivo data confirmed the anti-tumor efficiency of UROS gene therapy combined with 405nm-light exposure on subcutaneous prostatic tumors. Unfortunately, extensive dermotoxicity with skin bums and damage to the areas illuminated with 405nm-light was observed in all mice, regardless of UROS functionality (data not shown).

[0092] Alternative wavelengths for in vitro illumination of L7?O.S-inactivated PC3 cells

[0093] To reduce dermotoxicity and increase light penetrance while maintaining the efficacy of gene therapy, we applied longer light wavelengths at Q-band porphyrin absorption. We tested 530nm and 660nm wavelengths (data not shown). With 530nm, we obtained a scale-dose effect on UROS KO PC3 cells, with a partial effect from 24J / cm2(IC50) and a drastic reduction in cell count at 120J / cm2(corresponding to 20 minutes) (p<0.01). In contrast, illumination did not modify the cell count of WT PC3 cells (data not shown). The 660nm wavelength only induced a partial reduction of UROS KO PC3 cell count, even at the highest dose (230J / cm2, data not shown). We thus used 530nm light exposure thereafter as a compromise between penetrance and efficiency.

[0094] Because in vivo gene therapy cannot target all the tumor cells, a bystander effect would assist tumor regression. To evaluate the bystander effect of our strategy, we mixed 25% of UROS KO PC3 cells with 75% of WT PC3 cells in vitro and quantified the proportion of fluorescent cells three days after a single dose of 530nm light exposure (data not shown). A 50% drop in fluorescent cell count was observed, i.e. two-fold greater than the initial proportion of porphyrin cells, thus confirming the presence of a bystander effect (p=0.0022) (data not shown).

[0095] VROS inactivation combined with repeated 530nm light induces PC3 tumor regression in vivo

[0096] To evaluate the efficiency of UROS gene therapy combined with 530nm light in vivo, we subcutaneously grafted either WT or UROS KO PC3 cells in NSG mice to obtain one PC3 tumor per mouse. We next performed repeated 530nm illuminations i.e. three times a week, (day 0, day 2 and day 4) on PC3 WT and UROS KO PC3 tumors (Fig.lA), One control group of non-illuminated UROS KO PC3 tumors was also included in the experiment. We monitored tumor bioluminescence imaging (BLI) (data not shown) and tumor volumes by caliper. As observed in Fig.lB with BLI, UROS KO PC3 tumors were slightly smaller at the beginning of the experiment (BLI assay and volume measurement at day 0, Fig 1B / C, left panel), probably due to light exposure during injection. Repeated 530nm light for one week dramatically reduced tumor BLI signal intensities and tumor volumes. Tumors were undetectable at D7 to D10, even by BLI. At day 14, BLI intensities and volumes of illuminated UROS KO PC3 tumors were respectively 32- and 10-fold lower than those of non-illuminated ones. (Fig.lB / C, right panel, and illustrative Fig.lD), Moreover, no dermotoxicity was observed with repeated 530nm light exposure for one week (Fig.lD), Importantly, gene therapy associated with iterative 530nm light for one week doubled the survival of mice (34 days versus 17 days with UROS KO PC3 tumors) (Fig.lE),

[0097] VROS inactivation in PC3 cells combined with long-term repeated 530nm light increases mice survival

[0098] Because we observed a slight relapse around day 17 after three 530nm illumination sessions, we tried to extend the remission period by maintaining the same illumination protocol for five weeks (Fig.2A), Again, 530nm light was very efficient in blocking UROS KO PC3 tumor progression during the first two weeks (Fig.2B), Unfortunately, it did not avoid a secondary tumor relapse during week 3. To understand whether tumor relapses were due to i) the persistence and selection of UROS WT cells that are not light-sensitive or ii) a reduction in the efficiency of light after two weeks, we assessed the UROS molecular status of relapsing tumor cells by PCR. Sanger analysis revealed the persistence of a high proportion of UROS KO cells in illuminated tumors (data not shown). Therefore, at sacrifice, edited cells were still present. Histological analysis of tumors at sacrifice reveals a high modification of tumor structures (data not shown). After long-term light, tumors are disorganized and pauci-cellular. Importantly we observed a large peripheral reactional fibrosis. It suggests that regular external percutaneous long-term illumination did not reach the tumor cells, that could explain the relapse. Even though the tumors could not be completely eradicated, 530nm light exposure of UROS KO PC3 tumors dramatically increased survival compared to control groups without gene therapy and UROS KO PC3 cells without light. Survival was increased three-fold (42 days versus 14 days in the other conditions, Fi2. 2C)

[0099] Discussion:

[0100] We demonstrate that a CRISPR-Cas9-based gene therapy for UROS inactivation induces a high accumulation of porphyrins. As in congenital erythropoietic porphyria, these metabolic compounds are highly photosensitive. Lighting at 530nm combined with gene therapy led to the death of cancer cells in vitro without any side-effect, i.e. no mortality in non-transduced cells. Remarkably, we obtained in vivo tumor regression associated with a major increase in mice survival.

[0101] Owing to its high penetrance in tissues, a 630nm wavelength is often used for photodynamic therapy. However, it may prove non-optimal with only partial in vitro efficacy.15,16’17’18’19The 405nm wavelength, which is the Soret Band of porphyrins (optimal absorbance), was highly efficient but induced dermotoxicity and had very low tissue penetrance. As a compromise, we used a 530nm wavelength to avoid skin damage while maintaining medium penetrance and high in vitro efficacy. Using external illumination of the subcutaneous engrafted tumors (with the presence of the skin between light and the tumor), we obtained a satisfactory tumor response with tumor regression. While these results obtained with an external LED are promising, a therapeutic escape occurred. This may have been caused by a reduction in illumination penetrance due to remodeling of tumoral and peritumoral tissues. Thanks to the advent of interstitial lasers, it is now possible with endoscopic optical fibers to illuminate the interior of tumors, in direct contact with cancer cells.20’21Therefore, interstitial illumination combined with UROS gene therapy might lead to a longer response to treatment and could overcome the relapse. Moreover, human cancer cell xenografts require immunodeficient mice. We hypothesize that immune competent models obtained by using syngeneic mouse prostate tumors should allow the recruitment of the immune system, thus reinforcing the efficacy of our approach. Indeed, photodynamic therapy (PDT) is closely associated with strong immunogenic cell death (ICD) through the emission of damage-associated molecular patterns (DAMP) that attract and activate different immune cells.22

[0102] Our approach is an alternative to PDT, which uses an exogenous systemic photosensitizer (PS) drug such as aminolevulinic acid (ALA) to accumulate type IX-protoporphyrins (PpIX) or light-activated vascular occluding agents like TOOKAD®.23The two main limitations of PDT are an obstacle to its use in clinical practice for treating severe cancers and metastasis: (i) the low tumor specificity of PS accumulation in all tissues; and (ii) the low intra-tumoral concentration of PS in cancer cells after systemic administration of exogenous PS. ALA-PDT as a monotherapy often fails to achieve satisfactory clinical outcomes for treating cancer patients. Indeed, accumulated PpIX concentrations are low in tumors24,25and they induce the overexpression of inducible nitric oxide synthase and nitric oxide (NO),26,27resulting in a high rate of incomplete treatment response and disease relapse. Thanks to UROS gene therapy, we obtained high porphyrin concentrations in cancer cells without direct toxicity, i.e. without light, which is essential for preventing treatment side-effects and toxicity. Moreover, the type of porphyrins is different. The UROS deficit in CEP induces type-I porphyrin accumulation and severe mutilating skin lesions exposed to sunlight, compared to the ferrochelatase (FECH) deficit in erythropoietic protoporphyria (PPE) which leads to type-IX protoporphyrins associated with benign skin redness. These clinical data suggest that / / YAS'- edited cells accumulating cytosolic type-I porphyrins are more photo-cytotoxic than mitochondrial type-IX protoporphyrin and could be an attractive alternative to conventional PDT.

[0103] This pre-clinical proof-of-concept was achieved by using prostate cancer cells, since the prostate seems to be a relevant site for cancer gene therapy. It is accessible for the direct injection of intra-tumoral gene therapy and can be treated by illumination with optical fibers through endoscopes. Several gene-therapy clinical trials have already been approved for prostate cancer. Most of them target the tumor microenvironment and immune response. They include vaccine-based strategies,28,29and alteration of the immune microenvironment by IL- 230and CAR-T cells.31,32Protocols directly targeting prostate cancer cells are still rare and include suicide gene ,33>34>35’36oncolytic vectors,37suppressor gene activation (p53),38GLIPR1,39DDX5 mRNA targeting,40and vascular-targeted photodynamic therapy.41Despite some preclinical success, gene therapy is not yet routinely used for treating prostate cancer. Our strategy, mimicking a genetic disease to weaken the cancer cells combined with local tumor illumination, is a novel approach and could be a promising alternative. It allows a high specificity and efficacy in the lighted area without any peritumoral toxicity.

[0104] In this study, we ex vivo inactivated UROS before sub-cutaneous grafting as a proof of concept. In the future, the efficacy and safety of the approach should be tested after orthotopic prostatic grafting.42The in vivo injection of intra-prostatic gene therapy vectors or electroporation should allow specific porphyrin accumulation in the targeted organ. Unlike PS accumulation, the transgenic expression of CRISPR-Cas9 can be restricted to cancer cells under the control of a prostatic cancer promoter such as DD3 / PCA3.43,44Alternatively, it would be possible to use targeted vehicles with anti-PSMA, which is highly expressed on the surface of prostate cancer cells.45,46Nevertheless, type-I porphyrin accumulation alone is not toxic for cells and its combination with illumination is mandatory to be efficient. This is illustrated in CEP patients whose internal organs are not altered by porphyrin accumulation. This suggests the high level of safety of the approach. Even if gene therapy is not restricted to cancer cells, only the illuminated cells will die, thereby allowing temporal and spatial specificity. Altogether, the present findings show that CRISPR-Cas9 offers new insights into cancer gene therapy to invalidate the canonical metabolic pathway essential to cancer cell survival. Knowledge of the hereditary metabolic diseases would likely lead to new anti-cancer approaches.

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Claims

CLAIMS:

1. A method of treating cancer in a patient in need thereof comprising the steps of i) of increasing the content of photo-reactive porphyrins in cancer cells by invalidating the expression of UROS gene in said cancer cells and ii) light-activating the photo-reactive porphyrins in cancer cells to produce cytotoxic species, thereby inducing the death of cancer cells.

2. The method of claim 1 that comprises the steps of i) of increasing the content of photo- reactive porphyrins in cancer cells by editing the cancer cells to invalidate the expression of UROS gene in said cancer cells and ii) light-activating the photo-reactive porphyrins in cancer cells to produce cytotoxic species, thereby inducing the death of cancer cells.

3. The method of claim 2 that comprises the steps of administering to the patient a therapeutically effective amount of a DNA-targeting endonuclease that cleaves the genomic DNA of the cancer cells in at least one position located in UROS gene thereby invalidating the expression of said gene in said cancer cells.

4. The method of claim 4 wherein the DNA targeting endonuclease is a TALEN, ZFN, or a CRISPR-associated endonuclease.

5. The method of claim 4 that comprises the step of contacting the cancer cell with an effective amount of a CRISPR-associated endonuclease and with one or more guide RNA(s).

6. The method of claim 5 wherein the guide RNA is used for recruiting the CRISPR- associated endonuclease to the UROS gene and generating a DSB in exon 4.

7. The method of claim 6 wherein the guide RNA is used for recruiting the CRISPR- associated endonuclease to the UROS gene and generating a DSB between nucleotides at position 49 and 50, more particularly comprises a spacer sequence capable of annealing to the sequence as set forth in SEQ ID NO:2 (i.e. GCAGCAGAGTTATGTT).

8. The method of claim 7 wherein the guide RNA comprises the spacer sequence as set forth in SEQ ID NO:3 (i.e. GGAAGCAGCAGAGTTATGTT) for recruiting theCRISPR-associated endonuclease to the UROS gene and generating double-strand breaks between positions 49 and 50.

9. The method of claim 2 that comprises the steps of contacting the cancer cells with a gene-editing platform that consists of a (a) at least one base-editing enzyme and (b) least one guide RNA molecule for guiding the base-editing enzyme to at least one target sequence in the UROS gene and thereby repressing the expression of said gene.

10. The method of claim 2 that comprises the step of contacting the cancer cell with a geneediting platform that consists of (a) one prime editing enzyme and (b) one prime editing guide RNA (pegRNA) for guiding the prime editing enzyme to one target nucleic acid sequence in the UROS gene, thereby prime editing said region and subsequently repressing the expression of UROS in said cancer cells.

11. The method according to any one of claims 1 to 10 wherein the wave length ranges from 400nm to 700nm.

12. The method of claim 11 wherein the wavelength is about 530nm.

13. The method according to any one of claim 1 to 12 wherein the dose of light ranges from10J / cm2to 50J / cm2.

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