Description: 

The difficulties fighting a disease like cancer are twofold, firstly, almost every cancer is different in terms of underlying oncogenetics and secondly, it is very difficult to target cancer for destruction without also damaging healthy cell populations in the process. Herein is described a general method for the targeted destruction of all cell types with a distinguishable genetic difference from normal body cells.

First, genomic sequencing of healthy cell populations and abnormal cell populations is performed to determine the areas of genetic difference between them. Sequences which are present in abnormal cell populations are identified and CRISPR/Cas9-like guide RNA (gRNA) is created to target these differences present only in abnormal cells. The specific sequence of gRNA needed to target a CRISPR/Cas9-like protein to the genetic differences present only in abnormal cells is placed into a viral genome along with the genetic sequence needed to code for the production of a modified protein complex that is capable of complexing with guide RNA, identifying any target genetic sequences in the host cell’s genome, and then subsequently initiating a function if the target sequence is present (referred to henceforth as an if/then operator). The viral genome will also code for an endogenously-activated transcription factor, which is used to transcribe other chemically-activated transcription factors which will be used to control subfunctions within the viral plasmid which function to:

1. Increase the population of viral plasmids within a population of non-target breeding cells, whose purpose is to serve as a factory for viruses targeted to the target cells (cancer cell type) of interest.

2. Increase the overall number of copies of the viral genome in target cells by inducing production of viruses targeted to target cells. It will also induce the synthesis of the if/then operator protein and the gRNA needed to guide the if/then operator to complex with target genetic differences (if present) and then initiate subfunction 3.

1. Reduce the transcriptional drive of subfunction 3 by repressing the transcription factors responsible for driving subfunction 3.

2. Disables TF0 either directly or indirectly, which underpins the activity of subfunctions 1, 2, 4 and 5 or otherwise disabling the function. 

The function which drives all subfunctions besides subfunction 3 (which is referred to as function 0) serves to: 

1. Induce transcription of transcription factors 1, 2, 4 and 5, which, when chemically activated, serve to perform subfunctions 1, 2, 4 and 5 respectively. TF0 activity is needed to drive the overall function (including subfunction 3 indirectly via subfunction 2 unless subfunction 3 has enough transcriptional drive to achieve a positive feedback loop)

The only subfunction not directly driven by function 0 is subfunction 3, which functions to:

1. Induce cell death by transcribing cell-death-inducing genes inserted into the viral plasmid. Subfunction 3 initially relies on the if/then operator expressed in subfunction 2 to bind with target differences in the cell’s genome and then release a factor which causes expression of those genes which are transcribed by transcription factor 3. In addition to inducing cell death, subfunction 3 also serves to increase the amount of if/then operator targeted to selected differences, thus increasing its own overall transcriptional drive. The goal of this self-stimulation is to eventually produce a runaway positive feedback loop which kills cancer cells. 

The viral plasmid to be used to perform this function would likely be in the form of a stock plasmid containing insertion sites for custom cell death inducing factors as well as for the necessary viral components needed to produce viruses specific to the desired breeding and target cells. This way, the gene therapy can be flexibly tailored to kill cancers which resist death in a variety of ways and to target many kinds of target cells (ie skin, blood, liver, pancreas, brain, etc). In addition, custom guide RNA complementary to genetic sequences unique to cancer cell populations would be inserted to guide the if/then operator and a CRISPR/Cas9-like terminator. This custom plasmid would then be cultured in a breeding cell population to increase viral population before being tested for efficacy on biopsied tissue samples. Data would be collected during this stage to determine safety parameters for treatment and efficacy in destroying target cancer cells before injection and use in patients. 

 

Claims:

1. A viral plasmid which performs the following functions:

   1. Function:

      1. A primary transcription factor (TF0) which is responsible for transcribing the transcription factors responsible for the transcription of 4 different subfunctions:

   2. Subfunction 1:

      1. A secondary, exogenously-controlled transcription factor (TF1) responsible for inducing production of virus capable of infecting a breeding cell population (BCP) which is not of the cancer cell type.

   3. Subfunction 2:

      1. A secondary, exogenously-controlled transcription factor (TF2) responsible for inducing production of a virus capable of infecting a target cell population (TCP) which is of the cancer cell type.

      2. This transcription factor (TF2) also induces the transcription of an if/then operator which will increase transcriptional drive on transcription factor 3 (TF3) if it detects a target genetic sequence identified as being unique to cancer.

   4. Subfunction 3:

      1. A transcription factor (TF3) responsible for inducing cell death and increasing transcriptional drive on genes transcribed by TF3 in the case that an if/then operator detects genomic differences which are present in cancer cells but not in healthy cells. 

   5. Subfunction 4:

      1. A secondary, exogenously-controlled transcription factor (TF4) which is responsible for reducing transcriptional drive on genes transcribed by TF2 and TF3.

   6. Subfunction 5:

      1. A secondary, exogenously-controlled transcription factor (TF5) which is responsible for disabling the primary transcription factor or otherwise terminating the function.

2. A stock plasmid consisting of transcription factors TF0, TF1, TF2, TF3, TF4 and TF5, which transcribe:

1. TF1, TF2, TF4 and TF5.

2. A genome replicator.

3. A genome replicator and at least one if/then operator

4. At least one if/then operator.

5. A mechanism or mechanisms to decrease the transcriptional drive of TF2 and TF3.

6. A mechanism or mechanisms to terminate the function. 

1. A process by which healthy cells and cancer cells are biopsied, their genomes sequenced, analyzed for differences and guide ribonucleic acid (gRNA) is produced to target genetic differences present only in cancer cells. 

2. A process by which said custom gRNA targeted to genomic differences unique to cancer is placed into a stock viral plasmid to be transcribed by TF2 and TF3.

3. A process by which the genetic material needed to code for viral proteins which are capable of creating viruses which are able to infect a custom, primary, BCP and a custom, secondary, TCP are inserted into the stock plasmid to be transcribed by TF1 and TF2 respectively.

4. A process by which any custom, genetic material needed to code for cell death is inserted into the stock plasmid to be transcribed by TF3.

Abstract:

A customizable viral nucleic acid sequence designed to transcribe multiple forms of chemically-induced transcription factors which are responsible for transcribing certain gene sets (called subfunctions) in the event of an outside chemical stimulus. Subfunction 1 serves to induce viral replication of virus capable of infecting a breeding cell population, subfunction 2 serves to induce viral replication of virus capable of infecting a target cell population, subfunction 3 serves to probe its infected cell for any genetic sequences present in cancer but not in healthy cells. Subfunction 3 will initiate subfunction 4, which will express a host of cell death factors in response to a signal that cancer DNA is present in the cell. subfunction 5 reduces the drive on Subfunction 4, thus inhibiting cell death. Subfunction 6 will cause the viral genome to self-destruct. Custom gRNA can be made to target areas where a cancer cell’s genome differs from a healthy cell’s genome. Custom sequences are inserted into stock plasmid along with customizable cell death factors to be transcribed if cancer DNA is detected in the cell. The system uses a custom CRISPR-Cas9 protein designed to induce transcription of subfunction 4 in the event it binds with any of the custom genetic cancer sequences its gRNA has been targeted for.

Description: 

The difficulties fighting a disease like cancer are twofold, firstly, almost every cancer is different in terms of underlying oncogenetics and secondly, it is very difficult to target cancer for destruction without also damaging healthy cell populations in the process. Herein is described a general method for the targeted destruction of all cell types with a distinguishable genetic difference from normal body cells.

First, genomic sequencing of healthy cell populations and abnormal cell populations is performed to determine the areas of genetic difference between them. Sequences which are present in abnormal cell populations are identified and guide RNA (gRNA) for a CRISPR/Cas9-like complex is created to target these differences present only in abnormal cells. The specific sequence of gRNA needed to target a CRISPR/Cas9-like protein to the genetic differences present only in abnormal cells is placed into a viral genome along with the genetic sequence needed to code for the production of a modified protein complex that is capable of complexing with guide RNA, identifying any target genetic sequences in the host cell’s genome, and then subsequently initiating a function if the target sequence is present (referred to henceforth as an if/then operator). The viral genome will also code for an endogenously-activated transcription factor, which is used to transcribe other exogenously-activated transcription factors which will be used to control subfunctions within the viral DNA which function to:

1. Increase the population of viral copies within a population of non-target, breeding cells, whose purpose is to serve as a factory for viruses targeted to the target cells (cancer cell-type) of interest.

2. Increase the population of viral copies within a population of target cells by inducing production of viruses targeted to target cells. 

3. Induce the synthesis of the if/then operator protein and the gRNA needed to guide the if/then operator to complex with target genetic differences and then initiate subfunction 4 (if present).

1. Reduce the transcriptional drive of subfunction 4 by repressing the transcription factors responsible for driving subfunction 4.

2. Disables TF0 either directly or indirectly, which underpins the activity of subfunctions 1, 2, 3, 5 and 6 or otherwise disabling the function. 

The function which drives all subfunctions (which is referred to as function 0) serves to: 

1. Induce transcription of transcription factors 1, 2, 3, 5 and 6, which, when chemically activated, serve to perform subfunctions 1, 2, 3, 5 and 6 respectively. TF0 activity is needed to drive the overall function. All other subfunctions which can be performed by this viral DNA depend on the activity of the overall function.

The only subfunction not directly driven by function 0 is subfunction 4, which functions to:

1. Induce cell death by transcribing cell-death-inducing genes inserted into the viral DNA. Subfunction 4 initially relies on the if/then operator expressed in subfunction 2 to bind with target differences in the cell’s genome and then release a factor which causes expression of those genes which are transcribed by transcription factor 4. In addition to inducing cell death, subfunction 4 also serves to increase the amount of if/then operator targeted to selected differences, thus increasing its own overall transcriptional drive. The goal of this self-stimulation is to eventually produce a runaway positive feedback loop which kills cancer cells. 

The viral DNA to be used to perform this function would likely be in the form of stock, viral DNA containing insertion sites for custom cell death inducing factors as well as for the necessary viral components needed to produce viruses specific to the desired breeding and target cells. This way, the gene therapy can be flexibly tailored to kill cancers which resist death in a variety of ways and to target many kinds of target cells (ie skin, blood, liver, pancreas, brain, etc). In addition, custom guide RNA complementary to genetic sequences unique to cancer cell populations would be inserted to guide the if/then operator and a CRISPR/Cas9-like terminator. This custom viral DNA would then be cultured in a breeding cell population to increase viral population before being tested for efficacy on biopsied tissue samples. Data would be collected during this stage to determine safety parameters for treatment and efficacy in destroying target cancer cells before injection and use in patients.

Therapy would first involve exogenous activation of TF1 activity to increase the population of viral genomes in breeding cell populations. These cells would serve as the seed viral production source used to produce viruses designed for target tissue. It would then be followed by TF2 stimulation to increase the number of viruses targeted to target tissue. These would initially only come from breeding cells, but would eventually come from the target tissue in large amount until a high-enough viral load was reached to then initiate subfunction 3. TF3 stimulation would activate the many copies of viral DNA in target cells (and breeding cell tissue) to begin mass-producing the gRNA and if/then operators needed to probe the host cell’s DNA for any sequences unique to cancer. High amounts of viral DNA within cells would ensure that the conditional response to the presence of a target sequence would be robust enough to initiate subfunction 4, most likely by the cleavage of a linking domain connecting a TF4 domain and a Cas9 domain of a chimeric protein. The if/then operator would likely be a modified Cas9 protein designed to cleave an attached domain in response to pairing of gRNA and a target sequence (instead of the usual endonuclease-mediated cleavage of the target strand of DNA in response to pairing).

In any case, with the sustained stimulation of TF3, if the target genetic sequence was present, the if/then operator would initiate subfunction 4 which would induce death of cells in which a target genetic sequence was present. The drive on subfunction 4 also serves to increase its own drive, thereby pushing cell death into a positive feedback loop. Activation of subfunction 5 via stimulation of TF5 would serve to arrest the positive feedback loop of cell death in the case that non-cancerous cells are targeted. To reduce the probability of off-targeting, target sequence identification from genomic data would take into consideration the possibility of mutation in order to most effectively distance the gRNA sequence selected to be used to identify cells to kill from the rest of a healthy cell’s genome. Subfunction 4 also serves to inhibit subfunction 6, which is responsible for terminating the overall function and leaving the viral genome to be degraded by the cell, most likely via Cas9 targeted to the region coding for TF0. In either case, subfunction 6 serves to terminate the function and allow viral DNA to be degraded. At the end of one cycle of viral therapy, TF1 and TF6 are stimulated in the right amount to remove the viral load from target cell populations whilst ensuring enough production of viruses targeted to breeding cell tissue to maintain the breeding cell population in case further rounds of therapy are needed. The ability of subfunction 4 to inhibit the activity of subfunction 6 means that cells with target genetic differences present are less able to reduce their viral load, allowing viral load to accumulate in target cells across multiple rounds of therapy, thus increasing chances of cell death with time.

 

Claims:

1. A process by which:

   1. A primary transcription factor (TF0) transcribes the transcription factors, either directly or indirectly, which are responsible for the performance of multiple, unique operations, or otherwise, a function which enables the performance of subfunctions.

   2. A secondary, exogenously-controlled transcription factor (TF1) induces production of virus capable of infecting a breeding cell population (BCP) which is not of the cancer cell type, or otherwise, a subfunction (1) which functions to increase the population of viruses capable of infecting a cell type which is not the target cell type. 

   3. A secondary, exogenously-controlled transcription factor (TF2) induces production of a virus capable of infecting a target cell population (TCP) which is of the cancer cell type, or otherwise, a subfunction (2) which functions to increase the population of viruses capable of infecting a cell type which is the target cell type. 

   4. A secondary, exogenously-controlled transcription factor (TF3) induces the transcription of an if/then operator which increases transcriptional drive on TF4 if it detects a target genetic sequence identified as being unique to cancer in the genome of the host cell, or otherwise, a subfunction (3) which functions to probe the genetic composition of the host cell for the presence of genetic sequences unique to cancer. 

   5. A transcription factor (TF4) induces death of a host cell, increases transcriptional drive on genes transcribed by TF4 in the case that an if/then operator detects genetic sequences unique to cancer cells and decreases transcriptional drive on a set of genes responsible for destroying viral DNA, or otherwise, a subfunction (4) which functions to create a runaway positive feedback loop of cell death in response to the presence of genetic sequences unique to cancer and reduces the ability of subfunction 6 to terminate the function and destroy the viral DNA.

   6. A secondary, exogenously-controlled transcription factor (TF5) reduces transcriptional drive on genes transcribed by TF4, or otherwise, a subfunction (5) which functions to prevent runaway cell death.

   7. A secondary, exogenously-controlled transcription factor (TF6) which reduces the activity of the primary transcription factor and/or destroys the viral DNA, or otherwise, a subfunction (6) which terminates the function.

2. A process by which healthy cells and cancer cells are biopsied, their genomes sequenced, analyzed for differences and guide ribonucleic acid (gRNA) is produced to target genetic differences present only in cancer cells.