System, device and method for providing therapy or cure for cancer and other pathological states

Abstract

To provide a therapy of cancers, viral diseases as well as diseases caused by pathogenic organisms.SOLUTION: Provided is a method for treatment of pathologies caused by cells that have DNA sequences that differ from DNA of healthy human cells. The method utilizes targeting of the sequence differences and effects cleavage of the DNA which does not occur in the healthy genome, whereby the cells containing the targeted DNA are killed or inactivated. Preferred systems for effecting such DNA damage are CRISPR systems where a CAS cleaves DNA adjacent to a sequence recognized by the CRISPR.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to systems, devices and methods that allow for therapeutic intervention against various diseases, where the treatment involves the death of certain specifically defined cells, i.e. the present invention provides treatments for cancer, viral diseases and diseases caused by pathogens. [Background technology]

[0002] Millions of people die from cancer each year. Current treatments, such as surgery, chemotherapy and radiation therapy, are harsh and non-specific, killing many healthy cells along with the cancer cells they are intended to eliminate.

[0003] Although personalized cancer treatments have been attempted, most of these have not been proven effective against "any" cancer, since each cancer manifests uniquely in an individual, given the fact that cancer has a genetic etiology. A cancer in one patient's organ roughly behaves as a subset of a completely different disease in the same organ of another patient.

[0004] Furthermore, many viral infections and infections caused by live pathogens currently lack satisfactory treatments due to drug resistance or the unavailability of effective antibiotics that are not harmful to humans.

[0005] Thus, there is a need for cancer treatments that are customized to the particular cancer associated with an individual, and there is also a need for therapeutics that can specifically attack cells infected with viruses or pathogens that are difficult to target with existing antibiotics.

[0006] Several studies have proposed gene editing in cancer therapy, e.g., CRISPR-Cas9 screening of protein domains (Shi et al, Nature Biotechnology 2015), generation of novel cancer animal models (Mou et al. Genome Medicine 2015), transformation of known cancer-related genes and their conversion to healthy variants using viral vectors, and discovery of anti-cancer drug targets by using CRISPR-Cas9 in designing CAR-T immunotherapy (chimeric antigen receptor T cell immunotherapy).

[0007] In known technology, gene therapy in cancer treatment is used in in situ gene therapy protocols, which use viral vectors to transduce specific genes that increase systemic immunity against cancer (e.g., in situ immunomodulatory gene therapy in prostate cancer (The et al., Clin Med Res. 2006 Sep; 4(3): 218-227)). CAR-T cell immunotherapy works with the risks associated with immunotherapy and still poses risks and uncertainties for patients (e.g., autoimmune disorders, immunomodulatory side effects and death). Summary of the Invention

[0008] The present invention is based on the inventors' initial realization that using state-of-the-art, rapid and reliable genome sequencing and thus the identification of nucleic acid sequences that are unique to cells (such as cancer cells, virus-infected cells and pathogenic cells) compared to healthy cells of an individual, it is possible to design expression vectors that encode recognition and effector molecules that can selectively destroy or neutralize said cells.

[0009] According to findings from the Cancer Genome Project, most cancer cells carry more than 60 mutations. Because mutations vary between individuals, it has been difficult to identify the cause of these mutations in a specific type of cancer, but most of these mutations are unique to cancer cells and can therefore be differentiated when targeting these cells with specific therapeutic approaches. With bacteria and viruses, identifying unique sequences to target is less of an issue, but of course it is highly relevant to ensure that targeting specific viral or bacterial sequences does not result in unwanted off-target effects.

[0010] If a recognition molecule with very high reliability / fidelity can distinguish nucleic acid sequences from an individual's cancer from any of the nucleic acid sequences in the individual's normal cells, and if such specific recognition can be used to trigger the destruction or neutralization of the "cancer nucleic acid", then the process of providing an effector cancer treatment to an individual can be broken down into the following simple steps: 1) Identification and validation of nucleic acid sequences that are present in cancer cells but not in healthy cells, or at least that are present in only a small subset of healthy cells (sequences so identified are referred to herein as "cancer NA sequences"); 2) creating at least one expression vector encoding a recognition sequence complementary to the cancer NA sequence and encoding at least one effector cell capable of destroying or neutralizing nucleic acid containing the cancer NA sequence; 3) Administration of the expression vector to a patient suffering from cancer to effect targeted destruction or neutralization of a nucleic acid containing a cancer NA sequence.

[0011] The multiple mutations present in cancer cells versus normal cells provide multiple targets for the recognition sequence, so the effect achieved in destroying or neutralizing nucleic acids is comparable to the multiple DNA breaks that occur when treating cancer cells with radiotherapy. However, radiotherapy also causes the destruction of the DNA (and thus the destruction of) normal cells, and approaches that target cancer-specific nucleic acid sequences avoid off-target effects.

[0012] The inventors have realized that the exact same principle can be used to treat any type of disease in which a pathogen (a virus-infected cell, a bacterium, or a parasite) is the causative agent of the disease and contains a nucleic acid sequence that is different from any nucleic acid sequence of the infected individual. Such a distinguishable sequence is referred to herein as a pathogen NA sequence. The creation and administration to an individual of a suitable expression vector that encodes a recognition sequence complementary to the pathogen NA sequence and that encodes an effector molecule capable of destroying or neutralizing nucleic acid, including cancer NA sequences, can eradicate cells containing the target nucleic acid.

[0013] Thus, the present invention provides personalized genetic engineering based cancer treatments and genetic engineering treatments that target virally infected cells or pathogens.

[0014] Broadly speaking, the present invention provides a system comprising: interacting with the patient's material, or data derived from said material, in a predetermined response pattern; Providing designs for or synthesizing therapeutic engineered biomaterials; Following a predetermined response pattern based on computer means configured to: receiving input of the biological state of or data derived from samples, both disease-related and healthy, taken from the same patient, their genomic, transcriptomic, proteomic, gene expression, metabolomic and epigenomic data; Creating data in the design and / or sequence of biological materials and / or synthesizing biological materials based on predetermined patterns configured to be detrimental to the biological state of disease-related materials and not detrimental to healthy materials based on data collected from the patient.

[0015] In a first aspect, the present invention provides a method of treating a disease in an animal (such as a human) comprising inducing preferential death of cells which contain at least one DNA sequence which is not present, or is not present in substantial amounts, in healthy cells of said animal, comprising: 1) an effective amount of at least one expression vector, each of which comprises a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and a second nucleic acid sequence encoding a molecule that selectively destroys DNA comprising the at least one DNA sequence when the recognition molecule binds to the DNA; or 2) an effective amount of at least one first expression vector, each of which comprises a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one second expression vector comprising a second nucleic acid sequence encoding a molecule that selectively destroys DNA comprising the at least one DNA sequence when the recognition molecule binds to the DNA; or 3) an effective amount of at least one recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one molecule that selectively destroys DNA containing the at least one DNA sequence when the DNA is bound to the recognition molecule; or 4) an effective amount of at least one expression vector comprising a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one second molecule that selectively destroys the DNA comprising the at least one DNA sequence when the DNA binds to the recognition molecule; or 5) administering an effective amount of at least one recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one expression vector that includes a nucleic acid sequence that encodes a molecule that selectively destroys the DNA when the DNA containing the at least one DNA sequence is bound to the recognition molecule, The present invention relates to a method, wherein administration of said vector and / or molecule as defined in any of 1) to 5) results in the destruction of a nucleic acid molecule comprising said at least one DNA sequence to such an extent that said cell is killed or neutralized.

[0016] In a second aspect, the present invention relates to a method for designing and optionally preparing a therapeutic for treating a disease in an animal (such as a human), comprising: a) determining the sequence of DNA from cells or cell populations, including cells from malignant tissue, virus-infected cells and pathogenic cells; b) then comparing the DNA sequence information obtained in step a with DNA sequence information from healthy cells of said animal or from a completely sequenced healthy genome of said animal species; c) identifying DNA sequences from the cell or cell population that are not present in healthy cells of the animal; and d) Designing and optionally preparing a treatment that includes at least one of the following: 1) an expression vector comprising a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one DNA sequence identified in step c, and a second nucleic acid sequence encoding a molecule that selectively destroys the at least one DNA sequence identified in step c when the at least one DNA sequence is bound to the recognition molecule; or 2) a first expression vector comprising a first nucleic acid sequence encoding a molecule that specifically recognizes at least one DNA sequence identified in step c, and a second expression vector comprising a second nucleic acid sequence encoding a molecule that selectively destroys at least one DNA sequence identified in step c when the DNA sequence binds to the recognition molecule; or 3) an expression vector comprising a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one DNA sequence identified in step c and a molecule that selectively destroys at least one DNA sequence identified in step c when said recognition molecule binds to said DNA sequence; or 4) an expression vector comprising a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the DNA sequences identified in step c), and a molecule that selectively destroys at least one of the DNA sequences identified in step c when said recognition molecule binds to said DNA sequence; or 5) a recognition molecule that specifically recognizes at least one DNA sequence identified in step c) and a molecule that selectively destroys at least one DNA sequence identified in step c) when said DNA sequence is bound to said recognition molecule. Includes.

[0017] In a third aspect, the present invention relates to a computer or computer system for designing and optionally preparing the treatments described herein, comprising: i) a first computer memory or memory segment containing DNA sequence data from healthy cells of an animal or from a complete sequenced healthy genome of said animal species; ii) a second computer memory or memory segment containing DNA sequence data from disease-related cells; iii) executable code adapted to compare the DNA sequence data from the second computer memory with the entire set of DNA sequence data in the first memory to identify DNA sequences from the second computer memory that do not appear in the DNA sequence data in the first computer memory; iv) a third computer memory or memory segment suitable for containing DNA sequence data or a pointer to DNA sequence data identified by the computer as defined in iii; and v) executable code suitable for designing an expression vector based on 1) the DNA sequence data stored or represented in a third computer memory or memory segment, and 2) the sequence data in a nucleic acid encoding an effector molecule, and optionally, vi) a nucleic acid synthesizer suitable for synthesizing a DNA sequence designed by the executable code of v. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a flow chart illustrating the search and design algorithm, which is disclosed in detail in the first embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Recognition and effector molecules Much of the discussion and disclosure of the present invention focuses on identifying distinct nucleic acids from cancer / malignant cells. However, the present invention recognizes that current genome editing technologies, such as the CRISPR-Cas9 system, are unable to identify the specific nucleic acid sequences that are required to produce the desired results. Any It is understood that this is mainly based on the discovery that it provides the possibility of specifically targeting and editing nucleic acid sequences.See, for example, WO 2015 / 089465, which concerns the treatment of HBV infection by editing the genome of HBV-infected cells, and Hsin-Kai Liao et al, Nature Communications 6, Article no. 6413, which concerns gene-edited defense against HIV infection.

[0020] As described in the examples, the genome of malignant cells can be searched for sequences that can be targeted by recognition sequences, but ensure that healthy (normal) cells are not targeted.Indeed, the method in the first aspect of the present invention preferably results in the killing and neutralization of malignant cells in cancer patients, and it is also preferable that the recognition sequence used in this method does not recognize the self-DNA of animals in healthy (normal) cells.

[0021] The CRISPR system is the easiest in that it directs CRISPR-associated (Cas) proteins (such as Cas9 and any other effector molecules discussed in relation to CRISPR herein) to multiple gene targets by providing a guide RNA sequence complementary to the target site. Target sites in the CRISPR / Cas9 system can be found near most genomic loci; it only requires that the target sequence, which is compatible with the guide strand RNA, be followed by a protospacer adjacent motif (PAM) sequence. In Streptococcus pyogenes (Sp) Cas9, this is any nucleotide followed by a guanine pair (NGG). However, Francisella novicida Cas9 is designed to recognize the PAM YG (any pyrimidine followed by a guanine), and Francisella novicida Cpf1 recognizes the PAM TTN or YTN.

[0022] Therefore, the recognition molecule is preferably a nucleic acid, which recognizes said at least one DNA sequence, and the DNA sequence is targeted through base pairing.Typically, the recognition molecule is a crRNA, and the molecule that selectively destroys said at least one DNA sequence when it is combined with said recognition molecule is a CRISPR-associated protein (Cas) (i.e., nuclease).This Cas can be any suitable Cas, for example, Cas9, eSpCas9, SpCas9-HF and Cpf1.

[0023] The selection of therapeutic target sites and the selection of CRISPR systems may be further optimized by prioritizing targets via a predictive activity scorer for on- and off-target sites of the CRISPR system (such as, for example, SgRNAScorer 1.0 utilized at Harvard University).

[0024] In the present invention, cells targeted by the recognition sequence are preferably lethally damaged by destruction of multiple DNA molecules, although the therapeutic methods of the present invention are effective even when the targeted cells are merely "neutralized."

[0025] When using the CRISPR system in the specific recognition of various DNA sequences of malignant cells, the fidelity of recognition may be increased by using a recognition sequence that contains modifications in the backbone of the recognition sequence. For example, the stability of the duplex between the recognition sequence and its target DNA may be improved by using a recognition sequence that contains modified nucleotides. For example, locked nucleic acid (LNA) is mentioned; LNA-modified oligonucleotides (ribose modified) can form stable duplexes between relatively short complementary nucleic acid sequences, and in this manner, it is guaranteed that the risk of off-target effects can be minimized. Other possibilities are the use of phosphate backbone variants (such as phosphodiester and phosphorothioate internucleotide bond modifications), ribose variants (such as LNA, 2'OMe, 2'-fluoro RNA (2'F) and 2'MOE) and non-ribose backbone variants (such as PMO and PNA).

[0026] Because the modified nucleic acid is not an expression product, such an approach requires that the recognition sequence be provided by direct administration.

[0027] In the preferred embodiment of the present invention, the design and / or sequence of CRISPR system includes, but is not limited to, CRISPR-Cas9 system, CRISPR-eSpCas9 system, and SpCas9-HF system and CRISPR-Cpf1 system, or homologous recombination, RNA interference (RNAi), zinc finger nuclease (ZFN), transcription-activator like effector nuclease (TALEN) and other future methods to create precise, targeted changes to genome.This means, for example, that effector molecule can be selected from Cas9, eSpCas9, SpCas9-HF and Cpf1.Many of these exert minimal off-target effects, so the safety profile of the present invention mainly depends on the accurate identification of unique DNA sequences that are not found in normal cells.

[0028] An attractive feature of the present invention is that it can be tested in vitro before a patient undergoes treatment, which further improves safety. When planning to select recognition sequences, they can be administered to cancer cells isolated from a patient and to normal cells isolated from a patient. Only if the DNA-destroying effect is substantially restricted to cancer cells should the patient be subjected to the therapy. This method is particularly relevant for those embodiments disclosed herein that compare the DNA of cancer cells with a standard healthy genome, and as a measure of safety, this method ensures that the patient's normal cells do not accidentally contain one or more DNA targets that are considered to be specific to cancer.

[0029] Therefore, the embodiment of the present invention includes testing, optimizing and validating the compounded personalized therapy in healthy and diseased cells in vitro before administering the therapy in vivo.Prognostic results are measured by nucleotide resolution DNA double strand break mapping (e.g., direct in situ break labeling, enrichment with streptavidin and next generation sequencing (BLESS) etc.).Alternatively, single cell survival can be determined in vitro.

[0030] Bioinformatics search algorithms exist in the known art that, given a gene or an entire genome, can find all possible 20-base segments located near the PAM, rank them based on their uniqueness in the genome and other parameters, and generate a table of resulting guide RNAs (such as CHOPCHOP at Harvard or CRISPRscan at Yale). Thus, in this text, the discussion of DNA from malignant cells of a patient can be applied equally well to DNA from any pathogen or cell involved in a pathology, as long as said DNA is not identical to the DNA found in healthy cells of the individual being treated. If one wishes to target the specific DNA of cancer or other pathology-related cells, essentially only the choice of recognition sequence (e.g., its nucleic acid sequence) needs to be changed. Furthermore, the choice of the means to ensure that the recognition sequence enters the target cell must usually be optimized for the specific target cell. In summary, all disclosures in this application related to the design of recognition molecules in cancer can be used in a similar manner in other pathologies.

[0031] In some embodiments of the present invention, the given design and / or sequence is used in the CRISPR system or other future methods such as homologous recombination, RNA interference (RNAi), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and other methods to create precise, targeted changes to genomes.The currently preferred system is the CRISPR-Cas9 system, which has now developed into a versatile system for gene editing of eukaryotic (including human) genomes.

[0032] When using the CRISPR-Cas9 system, the only other requirement is that the sequence to be cleaved in the present invention is followed upstream or downstream by a protospacer adjacent motif (PAM) sequence (such as 5'-NGG-3').

[0033] As is clear from the claims, the treatment of the present invention can take many practical forms, it is only necessary to ensure that the target cells simultaneously harbor 1) a recognition molecule (such as at least one CRISPR having a DNA sequence complementary to a cancer or pathogen NA sequence) and an effector molecule (such as Cas9). Both molecules may be encoded on one and the same expression vector, or on separate expression vectors, or only one may be encoded on an expression vector and the other molecule may be directly administered, or both may be directly administered.

[0034] The preferred expression vector is adenovirus, adeno-associated virus or lentivirus, but any type of vector can be used that can provide recognition and effector molecules present in cells.Therefore, the methods generally applied in gene therapy that result in gene transfer into target cells are practical in the present invention.For example, when using longer and possibly modified nucleic acids that are not introduced into viral vectors but are instead of plasmids, it is attractive in the present invention to formulate them so that they are taken up by target cells.Liposome formulations are one possibility, but nucleic acids can also be directly modified, for example by binding with lipids such as cholesterol.

[0035] As will be appreciated, the preferred CRISPR-Cas9 system is usually used as a gene editing tool, rather than simply introducing breaks into target DNA. However, both gene editing and gene breakage techniques are useful in the present invention to kill or neutralize cells.

[0036] "Neutralization" means that the cell is inactivated by damaging its DNA, at least to the extent that it cannot proliferate (which in cancer means to halt the proliferation of the cancer) and / or to the extent that it cannot replicate its genome. Another possibility is that the cell becomes more immunogenic due to the appearance of immunogenic expression products and is therefore inactivated / dead as a result of the immune response. It is also possible that DNA damage induces at least partial reversion to a benign phenotype (meaning that the cell loses the ability to metastasize or to invade tissues). Finally, the cell is usually more sensitive to other anti-cancer treatments that may combine the principles of the present invention. However, it is preferred that the target cell dies as a result of the induced DNA breaks.

[0037] In one of the simplest embodiments, the therapeutic method of the present invention uses one or more recognition molecules (such as CRISPR) designed to target as many important chromosomes as possible in the target cell. The introduction of a sufficient number of breaks in chromosomal DNA then has the effect of making the cell non-viable, and as described above, this effect on the target cell is comparable to the effect of radiation therapy.

[0038] Frameshift mutations can also be site-specifically introduced by the method of the present invention (e.g., by using appropriately designed CRISPR-Cas9 or similar systems). Frameshifts are gene mutations caused by indels (insertion or deletion) of a number of nucleotides that are not divisible by 3 into DNA sequences. Due to the triplet nature of gene expression by codons, the insertion or deletion changes the reading frame (grouping of codons) and results in a completely different protein expression product compared to the unmutated gene. The earlier the insertion or deletion occurs in the sequence, the more the protein is changed.

[0039] When an indel frameshift occurs in one or more critical proteins of a target cell and the repair mechanisms are inhibited, as in cancer, the frameshift becomes incompatible with continued life in the cell.

[0040] A frameshift mutation is not the same as a single nucleotide polymorphism, which substitutes rather than inserts or deletes a nucleotide.

[0041] This technique is another way in which organisms can be destroyed.

[0042] When several sites are targeted simultaneously (eg, when excising larger fragments of an organism's genome, as is done in recombinant DNA engineering), larger effects can be induced through the lesions mentioned above.

[0043] Another way that can kill cells is to introduce one or several mutations that induce termination codons ("UAA", "UGA" or "UAG") in the sequence. The polypeptides produced will likely be abnormally short or abnormally long and non-functional.

[0044] In an interesting embodiment, the CRISPR-Cas9 system or a similar system is used to insert cell death genes or cell killing mechanisms into the target cells in order to efficiently and most quickly kill the cancer.

[0045] Simpler approaches rely on the introduction of one or more disruptions in the genome of the target cell, generally interfering with the cell's metabolism.

[0046] In another embodiment of the invention, the CRISPR-Cas9 or similar system used deletes one or more base pairs in the target cell genome to disrupt the cell's metabolism.

[0047] In another embodiment of the invention, the CRISPR-Cas9 or similar system modifies or inserts one or more genes in the genome of a target cell such that the cancer cells can be visually or electromagnetically detected (e.g., via fluorescent dyes) in the context of a surgical procedure.

[0048] In another embodiment, the CRISPR-Cas9 system is configured to generate single nucleotide polymorphisms that substitute rather than insert or delete nucleotides.

[0049] In another embodiment, several sites can be targeted simultaneously (e.g., to excise larger fragments of the organism's genome) to induce larger deleterious effects through the above-mentioned lesions, as is done in recombinant DNA engineering.

[0050] In another embodiment of the invention, the CRISPR-Cas9 system is configured to introduce one or more mutations that induce termination of a stop codon ("UAA", "UGA" or "UAG") within the sequence of the cancer cell.

[0051] As the claims make clear, the therapeutic method of the present invention finds a wide range of uses, so long as the genetic material is not present in the treated individual's own healthy cells, but is present in pathology-related cells.In addition to cancer, the present invention allows for the targeting of "difficult" bacterial and other infectious diseases (such as tuberculosis) or the specific targeting of bacteria or other pathogens that have developed drug resistance or are notoriously difficult to control / eradicate.Certain infectious diseases (such as malaria and many other parasitic diseases (such as schistosomiasis and Echinococcus multilocularis infections) and many viral diseases) currently have no effective treatment.

[0052] With regard to viral infections, the present invention may be used to target only those cells of an individual, which contain DNA from the virus (in these cases, the virus uses the biochemical machinery of the infected cell to produce viral proteins from DNA).Since the normal immunological defense against such viral infections is to induce the death of infected cells, the approach of the present disclosure is a simple approach that achieves the same end result in different ways.Target of interest is HIV infection, as well as HBV and HCV.

[0053] For other infectious diseases, the identification of the target DNA is not nearly as important due to the large differences between human DNA sequences and those found in bacteria, fungi and parasites. In these cases, the main challenge is to select an optimized method that results in the introduction of recognition and effector molecules into the target cells. In the case of bacteria, phages may be used as vectors, but other means (e.g., mycoviruses or nematode viruses) may be useful for introducing expression vectors into fungi and parasites.

[0054] Another embodiment of the first aspect of the invention integrates the active ingredient into a prophylactic or therapeutic depot (i.e., "vaccine") in the form of an injection, patch, pellet, or implant that administers a continuous dose of the active ingredient to the tissues, fluid compartments, or blood vessels of the animal to be treated, either prophylactically, before the onset of the disease, or after diagnosis. This embodiment requires the use of modern technology in drug deposition, ensuring sustained release.

[0055] Methods and Means for Identifying and Preparing Therapeutic Agents - Patent application In a preferred embodiment of the method and computer means of the present invention of the second aspect, both the genome of cancer or the genome of pathogens are compared with healthy genomes to find regions in the sequence that are different, which can lead to the design of the RNA part of CRISPR-Cas9 to selectively attack cancer cells or pathology-related cells and not attack healthy cells.This design of said RNA sequence can be achieved by collecting one or more samples from one or more healthy cells and one or more cancer cells, ensuring that the genome is representative of the healthy genome and the difference is representative of the cancer genome.Also, potentially, the difference exists in the majority of cancer cells, and therefore exists in multiple generations of cancer.This is due to the potentially rapid mutations that occur.

[0056] Thus, the methods and computational means of the second aspect can use the design and / or sequences provided in CRISPR-Cas9 systems (such as those detailed herein) or alternatively, homologous recombination, RNA interference (RNAi), zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) synthesis to create precise, targeted changes to the genome.

[0057] Generally, in the method of the present invention, which identifies useful sequences to target, this concerns the availability of information on the entire sequence in the normal cells of the individual to be treated.However, this can be omitted: this is not the case with a genome that has already been sequenced, but instead, a genome that has already been sequenced (healthy) can be used, which is slightly less safe, because of the risk that the individual to be treated may contain sequences in common with the disease sequence.However, as shown herein, an initial safety assessment can be carried out in vitro by treating malignant cells and healthy cells from the individual to be treated with a combination of a recognition molecule and a molecule that destroys the target DNA sequence when the target DNA sequence binds to the recognition molecule.

[0058] When starting to sequence disease-related cellular DNA, it may take some time before the whole genome is sequenced, but during the process (perhaps even at a very early time), useful target sequences can be continuously identified.As each sequence is identified, it is added to a table of potential target sequences, which can be ranked and re-ranked against the sequences already included and the sequences that are continuously included.The criteria used to rank sequences include evaluation of the presence of PAM, the presence of sequences in important chromosomes, etc. (see below).

[0059] It may also be convenient to compare the cancer DNA sequence to one or more fully sequenced normal genomes and search for differences relative to highly conserved genes in the prior sequenced genome.

[0060] In addition, a table of about 200 "common" genetic mutations that are associated with and specific for many cancers is already known. When such mutations are identified as part of the identification process, these sequences are first ranked as a standard in the later preparation of targeted cancer therapeutics, since it is known that these sequences are cancer specific, and the inclusion of recognition sequences that target such cancer specific sequences is a safe option.

[0061] In one embodiment of the invention, the output of the system is incorporated into an adenovirus (ie, adeno-associated virus) or lentivirus as a therapeutic vector.

[0062] In one embodiment of the present invention, the computer means is configured to search and detect sequences of the cancer genome that differ from the healthy genome having 1-20 nucleotides at the same position of the cancer genome and the healthy genome, and is configured to find such sites with a protospacer adjacent motif (PAM) sequence (5'-NGG-3') downstream or upstream. If the system finds multiple such sites, it is configured to rank the found sequences based on the most different sites or based on a database of knowledge on the importance of the gene of interest. The system is configured to at least one of the following: 1) rank the found sites with the most difference between the healthy site and the cancer / pathogen-associated site highest; 2) rank the found sites whose use is known to have the highest impact based on a database of predetermined knowledge; and 3) indicate the location of these found sites. 4) Create a treatment plan and / or synthesize a treatment based on the discovered sites by copying the discovered sites into an RNA sequence, where the synthesized treatment can be included in a CRISPR-Cas9 system and is a prescription in a unit that specifically targets cancer cells and not healthy cells. 5) Generate several outputs (such as several CRISPR-Cas9 systems) that attack several different points in the target cancer material (e.g., multiple points in the cancer genome) with the goal of destroying larger parts of the cancer material. When attacking several points in the cancer cell simultaneously, the goal is to cut out a larger part of the genome and cause more damage.

[0063] In one embodiment of the invention, the genetic input is configured to extract cancer genetic material from a patient's blood sample using liquid biopsy.

[0064] In another embodiment of the present invention, a computer means obtains this database of differences between genomes and identifies differences that include, are adjacent to, or are close to the PAM site (e.g., differences between two genomes that are 1-20 nucleotides away from the PAM site). Essentially, even a few mismatches between normal DNA sequences and DNA sequences found in cancer can be used as targets. In particularly interesting embodiments where longer mismatches are identified, multiple, partially overlapping recognition sequences can be designed to allow multiple possible cleavages in cancer cell DNA.

[0065] In one embodiment, the machine can receive one or more samples of healthy cells and one or more samples of diseased cells and ensure the identification of sites likely to represent segments of abnormal DNA representative of different generations of diseased cells.

[0066] In order to improve existing cancer treatments and to enable safe vectors with high specificity for cancer only, the present invention provides a system that can collect several data samples that consider cancerous and non-cancerous substances, taking into account the likelihood that the data represents each type of substance, thus improving the level of safety and specificity.

[0067] Due to its configuration and computational means, the system allows for highly specific cancer treatment, which takes into account the identity of the patient's healthy cells as well as the makeup of the patient's cancer cells.

[0068] In an embodiment of the invention, the computer means is configured to read a digital document comprising at least genomic data and to create a digital document comprising at least genomic data.

[0069] Due to the system's computational means and ability to receive and interpret patient data, the device enables cancer treatments that take into account the specific genome of an individual's actual cancer in the design of targeted, genetically engineered treatments.

[0070] In another embodiment, computer system is configured to generate the information that determines the treatment vector and its design based on the genome of each patient.It just needs to pre-program the computer with the sequence information of one or more expression vectors, into which the sequence that codes for recognition molecule and effector molecule can be inserted.Optionally, computer system can be linked to nucleic acid synthesizer, or computer system can deliver synthetic input to nucleic acid synthesizer, which can generate the expression vector useful for the present invention.

[0071] The device facilitates a secure means by which computers interact with a database of patient-based data and insights that consider optimal strategies to damage cancer cells.

[0072] In one embodiment, the database contains information about and about gene loci and their gene products that are known to be common in humans.

[0073] As the claims make clear, the presently described computer system and associated methods are useful in a method of providing a treatment or cure for cancer, said method comprising interacting with a patient's substance or data derived from said substance in a predetermined response pattern; Providing designs for or synthesizing therapeutic engineered biomaterials; Following a predetermined response pattern based on a computational methodology consisting of; receiving input of the biological state of or data derived from samples taken from the same patient, its genome, transcriptome, proteome, gene expression, metabolome and epigenome, both from non-cancerous and cancerous materials; Creating data in the design and / or sequence of the biological material and / or synthesizing the biological material based on a predetermined pattern configured to damage the biological state of the cancerous material and not damage the non-cancerous material based on the collected patient data.

[0074] As is evident from the above paragraphs, these methods may further utilise one or more aspects of the systems referred to above.

[0075] Thus, although the preferred embodiment of the present invention has been described in detail, it is understood, and will be apparent to those skilled in the art, that many modifications not illustrated in the detailed description of the present invention may be made without changing the concepts and principles of the present invention embodied herein. It is also understood that many embodiments incorporating only parts of the preferred embodiment are possible without changing, for their part, the concepts and principles of the present invention embodied herein. Thus, the present embodiment is to be considered in all respects as illustrative and / or descriptive, and not restrictive, the scope of the present invention being set forth in the appended claims, and all alternative embodiments and changes to the embodiments shown herein that come within the meaning and scope of the equivalents of the appended claims are therefore embraced herein. EXAMPLES

[0076] In the following, embodiments of the invention are explained in more detail with reference to the figures: Figure 1 shows a step-by-step diagram of the calculation.

[0077] Example 1 Bioinformatics proof-of-concept experiment FIG. 1 is a step-by-step diagram of the calculations, which are given in detail below. The input parameters for the computer system are the DNA sequences from cell populations representing healthy tissues and the DNA sequences from cell populations representing malignant tissues in this experiment. The following algorithm may be provided with the input parameters from virus-infected cells and pathogenic cells. The sequencing data can be whole genome, partial and / or deep DNA or RNA sequencing.

[0078] First, the PAM sequence of healthy or target cells is identified using KMP (Knuth-Morris-Pratt) string search method. The PAM sequence in this experiment is indicative of the future use of CRISPR-Cas9, and therefore was 5'-NGG-3'. The PAM sequence, which includes DNA nucleotides with the length of the desired complementary recognition molecule (e.g., CRISPR RNA) before and after, is saved in the form of a table for later comparison. Trials were performed using a custom algorithm created in the BioPython open source project and samples of chromosome 2 from healthy and cancer genomes from the same individual found in the AWS1000 Genome Project database (ref|NW_004929308.1).

[0079] The sample sequences of chromosome 2 are then inserted into their respective set (or bag) data structures. Sets allow for overlapping values ​​so as not to ignore more occurring sequences, and partial matching analysis can be performed. The set data structure is used because its constant running time is O(1), which is important for larger DNA sequences.

[0080] As a third step, the two respective sets are subtracted (or subtracted) to produce a third set of results. The subtraction results in a set of complementary recognition molecules that are unique to the targeted cancer cells and not present in healthy cells. In an experiment conducted with 230 million nucleotides, including overlaps, 8 million potential targets were identified, with PAMs occurring at various positions in the target sequence.

[0081] Further analysis may then be performed on the resulting set of sequences unique to cancer or healthy cells, as appropriate, in ranking their uniqueness, ranking target sequences that correlate with targets within sequences known to correspond to portions of the exome, ranking targets based on their predicted activity scorer (e.g., via SgRNAScorer1.0), ranking further optimized targets based on their usefulness (e.g., in designing LNAs), ranking based on their feasibility of being expressed into a specific vector (e.g., an adeno-associated virus or another format vector that may provide for the presence of recognition and effector molecules in the cell), etc.

Claims

1. 1. A method of treating a disease in an animal (such as a human) comprising inducing preferential death of cells that contain at least one DNA sequence that is absent, or absent in substantial amounts, in healthy cells of said animal; 1) an effective amount of at least one expression vector, each of which comprises a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and a second nucleic acid sequence encoding a molecule that selectively destroys DNA comprising the at least one DNA sequence when the DNA binds to the recognition molecule; or 2) an effective amount of at least one first expression vector, each of which comprises a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one second expression vector comprising a second nucleic acid sequence encoding a molecule that selectively destroys DNA comprising the at least one DNA sequence when the DNA comprises the at least one DNA sequence and the recognition molecule binds to the at least one DNA sequence; or 3) an effective amount of at least one recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one molecule that selectively destroys DNA containing the at least one DNA sequence when the DNA binds to the recognition molecule; or 4) an effective amount of at least one expression vector comprising a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one second molecule that selectively destroys the at least one DNA sequence when the at least one DNA sequence binds to the recognition molecule; or 5) administering an effective amount of at least one recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one expression vector that includes a nucleic acid sequence that encodes a molecule that selectively destroys the DNA when the DNA containing the at least one DNA sequence is bound to the recognition molecule, A method according to any one of claims 1) to 5), wherein administration of said vector and / or molecule results in the destruction of a nucleic acid molecule comprising said at least one DNA sequence to such an extent that said cell is killed or neutralized.

2. 2. The method of claim 1, wherein the animal is not exposed to any recognition molecule that recognizes the animal's own DNA in healthy cells. (Claim 2) 2. The method of claim 1, wherein the disease is cancer and the cells to be killed or neutralized are malignant cells.

3. 2. The method of claim 1, wherein the disease is an infectious disease selected from a bacterial infection, a fungal infection and a parasitic infection, and the cells to be killed or neutralized are cells from a pathogen.

4. 2. The method of claim 1, wherein the disease is a viral infection, the animal's cells contain DNA from an infectious virus, and the cells to be killed or neutralized are cells containing DNA from the infectious virus.

5. The method according to any one of claims 1 to 4, wherein the recognition molecule is a nucleic acid and recognizes said at least one DNA sequence via base pairing.

6. 6. The method of claim 5, wherein the recognition molecule is a crRNA and the molecule that selectively destroys the at least one DNA sequence when the at least one DNA sequence binds to the recognition molecule is a CRISPR-associated protein (Cas).

7. 7. The method of claim 6, wherein the Cas is one of Cas9, eSpCas9, SpCas9-HF and Cpf1.

8. 1. A method for designing and optionally preparing a therapeutic in the treatment of a disease in an animal (such as a human), comprising: a) determining the sequence of DNA from cells or cell populations, including cells from malignant tissue, virus-infected cells, and cells of pathogens; b) then comparing the DNA sequence information obtained in step a with DNA sequence information from healthy cells of said animal or from a completely sequenced healthy genome of said animal species; c) identifying DNA sequences from the cell or cell population that are not present in healthy cells of the animal; and d) designing and optionally preparing a therapeutic regimen, comprising at least one of the following: 1) an expression vector comprising a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one DNA sequence identified in step c) and a second nucleic acid sequence encoding a molecule that selectively destroys the at least one DNA sequence identified in step c) when the at least one DNA sequence is bound to the recognition molecule; or 2) a first expression vector comprising a first nucleic acid sequence encoding a molecule that specifically recognizes the at least one DNA sequence identified in step c, and a second expression vector comprising a second nucleic acid sequence encoding a molecule that selectively destroys the at least one DNA sequence identified in step c when the at least one DNA sequence binds to the recognition molecule; or 3) an expression vector comprising a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one DNA sequence identified in step c) and a molecule that selectively destroys the at least one DNA sequence identified in step c) when the at least one DNA sequence is bound to the recognition molecule; or 4) an expression vector comprising a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one DNA sequence identified in step c) and a molecule that selectively destroys the at least one DNA sequence identified in step c) when the DNA sequence binds to the recognition molecule; or 5) a recognition molecule that specifically recognizes at least one DNA sequence identified in step c) and a molecule that selectively destroys the at least one DNA sequence identified in step c) when the DNA sequence is bound to the recognition molecule; The method includes:

9. The method of claim 8, wherein step c also comprises ranking the identified sequences.

10. A computer or computer system for designing and optionally preparing a treatment regimen as defined in claim 8 or 9, comprising: i) a first computer memory or memory segment containing DNA sequence data from healthy cells of an animal or from a complete sequenced healthy genome of said animal species; ii) a second computer memory or memory segment containing DNA sequence data from disease-related cells; iii) executable code adapted to compare the DNA sequence data from the second computer memory with the entire set of DNA sequence data in the first memory and identify DNA sequences from the second computer memory that do not appear in the DNA sequence data in the first computer memory; iv) a third computer memory or memory segment suitable for containing the DNA sequence data or a pointer to the DNA sequence data identified by the computer defined in iii; and v) executable code suitable for designing an expression vector based on 1) the DNA sequence data stored or represented in the third computer memory or memory segment, and 2) the sequence data in the nucleic acid encoding the effector molecule, and optionally vi) A computer or computer system, including a nucleic acid synthesizer, suitable for synthesizing a DNA sequence designed by the executable code of v.

11. 11. The computer or computer system of claim 10 configured to use predetermined designs and / or sequences in the synthesis of a CRISPR-Cas9 system (such as a CRISPR-Cas9 system comprising Cas9, eSpCas9, SpCas9-HF or Cpf1, or homologous recombination, RNA interference (RNAi), zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) that create precise, targeted changes to a genome).

12. A computer or computer system according to claim 10 or 11, adapted to carry out the method according to steps b to d of claim 9.

13. 1. A composition for use in a method of treating a disease in an animal (such as a human), the method comprising inducing preferential killing of cells that contain at least one DNA sequence that is absent, or absent in substantial amounts, in healthy cells of said animal, said composition comprising: 1) an effective amount of at least one expression vector, each of which comprises a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and a second nucleic acid sequence encoding a molecule that selectively destroys DNA comprising the at least one DNA sequence when the DNA binds to the recognition molecule; or 2) an effective amount of at least one first expression vector, each of which comprises a first nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one second expression vector comprising a second nucleic acid sequence encoding a molecule that selectively destroys DNA comprising the at least one DNA sequence when the DNA comprises the at least one DNA sequence and the recognition molecule binds to the at least one DNA sequence; or 3) an effective amount of at least one recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one molecule that selectively destroys DNA containing the at least one DNA sequence when the DNA binds to the recognition molecule; or 4) an effective amount of at least one expression vector containing a nucleic acid sequence encoding a recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one second molecule that selectively destroys the DNA containing the at least one DNA sequence when the DNA binds to the recognition molecule; or 5) an effective amount of at least one recognition molecule that specifically recognizes at least one of the at least one DNA sequences, and an effective amount of at least one expression vector that includes a nucleic acid sequence that encodes a molecule that selectively destroys the DNA when the DNA containing the at least one DNA sequence is bound to the recognition molecule; A composition, wherein administration of said vector and / or molecule as defined in any of 1) to 5) results in the destruction of a nucleic acid sequence comprising said at least one DNA sequence to the extent that said cell is killed or neutralized.

14. A composition for use according to claim 13, wherein the method is as defined in any one of claims 2 to 7.

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