Molecular Artificial Neurons, or, “Molecular Stuxnet”

A Modular Nucleotide System for the Treatment of Disease via Threshold-based Conditional Genomic Logic

US Patent Application — Written 2 October, 2013

This process is a novel, precise, and personalized medical treatment paradigm for cancer, microbes, and gene therapy.

Current state-of-the-art medical therapy for cellular diseases involves the use of pharmaceutical molecules which distribute through the human body via principles of pharmacology, physiology, and 3D structural biology. Many pharmaceuticals closely mimic the molecules used for normal physiological processes and therefore fit tightly into the same molecular machines, exerting their effect by manipulation of the dynamic movement of the machine’s 3D atomic mechanical structure. Side effects occur when the drug molecule exerts effects upon processes besides the therapeutic target. Charged molecules cannot pass easily across cellular lipid membranes, and thus drug absorption depends heavily on acid/base conditions at the absorption site. Many pharmaceuticals travel through cellular pores to gain entry to cells, and cells often evolve machines in vivo that expel or degrade these drugs, leading to resistance. One way to avoid the evolution of resistance in a cellular population, such as in cancer, is to kill all of the cells at once, so that they cannot continue evolving. Thus, this therapy is designed to avoid side effects and the evolution of resistance by specifically targeting desired cellular subpopulations in the patient’s body for genome engineering by precise verification of the digital information in their DNA or RNA nucleotide sequences and conditional assembly of nanoscale effectors within target cells by using the cell’s own manufacturing processes. These medical nanosystems must be carried into the cells by a vector, typically a lipid or polymer nanoparticle or viral protein capsid. As the human immune system is evolved to repel unknown protein antigens, nanopolymers may be preferable. It is important that medical nanosystems are not designed to replicate, as internal self-replication may cause sustained or exponential growth in nanosystem concentration which can harm patients.

A sterile, sealed device will receive patient samples, analyze cells from those samples, and use the data generated to guide the manufacture of a large number of biocompatible nanovectors, with each vector containing an identical set of modular and target-specific nucleotide therapeutics. These vectors will insert their DNA, RNA or nucleotide derivative (Nd) molecules into cells. RNA is an ideal substrate for this system, because it is an informational biopolymer which can form base pairs with DNA and RNA, execute enzymatic activities, or be translated into protein machines. The modular RNA molecules will conditionally assemble effector biomolecules only within cells bearing a sufficient threshold of correspondence to the predetermined target genomic sequence and will not assemble this mechanism within non-target cells. Effector molecules, once assembled within target cells, can exert a range of effects as needed to treat pathology, such as inducing cancer/microbial cell death, removing or destroying viral/unwanted code sequences, inserting desired gene therapies, or dynamically modulating gene expression patterns. By only exerting its effect on target cells while sparing normal cells, this therapeutic strategy may prevent or reduce patient side effects, improve patient quality of life, and improve specific patient outcomes versus chemical therapeutics which affect undesired cells in the patient’s body in an imprecise manner.

1) Sample Prep and Sequencing: The device first receives patient tissue, robotically isolates single cells via optical identification and pipetting or via chemical dissolution of extracellular structures and microfluidics, and performs multiplexed DNA sequencing on cells individually, e.g. tumor biopsy, blood culture, swab, circulating tumor cell lab-on-a- chip. Further samples of normal patient tissue can be sequenced to provide additional information on normal patient cell genome sequences.

2) Algorithms: Next, the data from this set of single cell genome sequences, each around three billion nucleotides long, will be processed via two algorithms:

2A) Classifier System: This is a bayesian machine-learning binary classification algorithm which will classify each cell as being a member of the target or non-target group by comparing each cell’s nucleotide sequence to other patient sequences and to a database of known pathogenic mutations, reference genome sequences, and cellular interaction networks. The output of this algorithm will be two sets of single-cell genome sequences: target and non-target.

2B) Target Substring Identifier System: This system will identify substrings of consecutive nucleotides which are only found in the group of target cells and not in normal cells. This is a multiple-sequence common-substring problem akin to 2013-era plagiarism detection algorithms. The output of this algorithm will be a set of substrings of nucleotides which are only found within target cells and not within normal cells.

3) Nanovector/Nucleotide System: Next, the output from algorithm B will be transmitted to a nanovector and nucleotide synthesizer device. Operators, typically medical professionals, will interface with this device via a touchscreen panel or attached computer terminal to direct the assembly of personalized therapies on a per-patient basis as needed to correct patient-specific pathology. This device will assemble biocompatible lipid, protein, or polymer based nanovectors containing modular nucleotide therapeutics by synthesizing groups of oligonucleotides into the inner core of these vectors. Vector surfaces may be studded with biomolecules designed to bind to cell-surface markers to gain access to the area of interest, but due to the inherent specificity of combinatorial logic, this may not be necessary for treatment success. Once built, vectors will be stored in climate-controlled and precisely chemically maintained reservoirs until the patient is prepared to receive treatment. Reservoirs will be labeled with time of manufacture and patient initials so as to prevent medical errors. Each therapeutic molecule will contain three subdomains:

3A) Specificity Subdomain: This is a section of nucleotides which will base-pair to a substring of target genetic code and induce a conformational change in subdomain B upon target substring recognition. Since genomes change over time, a large variety of these subdomains may be inserted into each nanovector to cover the range of substrings which may be found in the target cells.

3B) Cleavage Subdomain: This is a section of nucleotides which will change shape or conformation upon the binding of subdomain A to its target substring. This change in shape will then permit this subdomain to perform an enzymatic function and cut itself, thus releasing subdomain C into the cell. A modified cis- cleaving hammerhead ribozyme sequence would be a potential example.

3C) Effector Subdomain: This is a section of nucleotides which will be released into the cell upon the activation of subdomain B by subdomain A. These nucleotides will then either perform genetic interference, translate into single or multiple subunit protein machines within the cell, or assemble to form a ribozyme RNA machine. Akin to neuronal action potential thresholds, once a sufficient threshold of effector subdomains is released into the cell, these effector subdomains will assemble into an effector or will exert separate effects which combine to produce the desired net change in cellular function. Threshold-based activation allows the therapy to maintain effectiveness in a setting of genomic heterogeneity, where target cells mutate individually and lose various target substrings yet remain similar overall.

3A.1) Cell Death: To conditionally destroy pathologic cells such as in cancer, the effector molecule will target conserved cellular functions. One method is to interfere with transcription and translation of DNA into molecular machines via RNAP, RNA, or Ribosome inhibition. Cells which cannot execute this function will invariably die due to inability to manufacture necessary internal components. Further, all cells contain cellular membranes, and so a mechanical attack on these membranes launched from within may also be effective in destroying target cells of all forms. Finally, an inhibition of the glycolytic pathway could prevent glucose metabolism, a common metabolic pathway in many pathogenically relevant cells. By using an effector which is able to kill many different forms of cell, this could be used for bacteria, cancer, parasites, and virally infected cells.

3A.2) Viral Removal: Some viruses, such as HIV, insert their genome sequence into the host cell’s genome. Conditionally assembled effector molecules within viral-genome-hosting cells could remove or destroy viral DNA, RNA, or protein without incidental damage to normal cellular functions as experienced by patients treated with immunomodulatory or fraudulent nucleotide antiviral therapeutics.

3A.3) Gene Insertion: For conditional insertion of desired genes within targeted patient cellular subpopulations only, conditionally assembled effector molecules could perform a function of reverse transcription and DNA integration in order to permit targeted gene insertion in specific portions of the body and not in others. Localization of this targeting could be performed by conditioning the assembly of the effector molecule on the dynamical expression pattern of mRNA corresponding to the target cellular differentiation state.

3A.4) Gene Expression Regulation: Conditionally released effector molecules can be used to change a range of cellular expression patterns and thus produce desired changes in cellular phenotype. Effectors can inhibit various protein machines, interfere with or promote transcription of disease-relevant genes, and could conditionally target various epigenetic phenomena.

4) Delivery to the Patient: Manufactured vectors will be given to the patient in a procedural setting. If the setting is a tumor excision surgery, vector particles can be suspended in a gel which could be used to fill the tumor cavity to prevent relapse by killing any tumor cells which attempt to regrow into the area. Further, vectors could be injected into the patient’s bloodstream in order to neutralize circulating tumor cells and reach metastatic foci. Vector insertion could be performed via endovascular methods, such as release of vectors upstream of the blood supply of a tumor or infected area. Direct percutaneous injection of vectors into pathologic sites may increase effectiveness, and depth of injection into the walls of the tumor cavity may be necessary to target migratory cancer stem cells. If cellular death is the goal of the therapy, caretakers must control and mitigate any subsequent sudden release of intracellular components and ions, such as potassium, urate, phosphorus, and calcium. This pathophysiological response to sudden large amounts of cell death is often referred to as the tumor lysis syndrome, and can produce DIC or ARDS if improperly managed. Dialysis may be necessary. Filtration of vector particles may be necessary in patients with disruptions in vector excretion, which may be via kidney or liver depending on vector construction.

5) Maintenance of the Device: The device will be designed to maintain sterile internal conditions, reduce cost and lessen practitioner workload as much as possible by implementing self-cleaning functions and reusing materials unless contamination has occurred. Occasional replenishment of raw materials in the form of sequencing reagents, nanovector ingredients, RNA/RNAd nucleotides, cleaning supplies, and replacement parts will be necessary, but the device will use refillable cartridges to reduce workload and waste.

6) Components of the Device to Perform the Process:

• Outer Casing + Sterile/Microbe-resistant Seal
• Air Intake and HEPA Filter System
• Patient Sample Intake System
• Robotic single-cell isolation System (optical/mechanical or chemical/microfluidic)
• Single-cell DNA sequencing System (multiplexed for high throughput)
• Tamper-proof construction and biometric scanning system.
• Encryption and firewalls to prevent unauthorized remote access.
• System to reject the production of unsafe or hazardous nanosystems.
• Computer Processing Unit, Display, Interaction System (keyboard, mouse, touch)
• Nanoparticle & RNA(d) Nucleotide synthesizer System
• Climate-Controlled Reservoir System — Labeled w/ Pt Initials to prevent errors
• Cleaning, Rinsing, UV, Heat based sterilization to prevent cross-contamination

7) Categories:

G06F 19/24 — Bioinformatics

Refs:
1. An unprecedented look at stuxnet, the world’s first digital weapon, https://www.wired.com/2014/11/countdown-to-zero-day-stuxnet/
by Zetter

2. Intratumor Heterogeneity & Branched Evolution Revealed by Multiregion Sequencing, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878653/
by Gerlinger et al

3. Multi-input RNAi-based logic circuit for identification of specific cancer cells, https://www.ncbi.nlm.nih.gov/pubmed/21885784,
by Xie, Wroblewska, Prochazka, Weiss, and Benenson

4. A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads,
https://pdfs.semanticscholar.org/0d74/249668b64e2c0f4a7c5da10762000a46425e.pdf by Douglas, Bachelet, and Church

5. Chemistry and Biology of Self Cleaving Ribozymes,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630146/
by Jiminez, Polanco, and Luptak

6. Threshold Potential, https://en.wikipedia.org/wiki/Threshold_potential

Copyright 2018 Bion Howard & bitpharma.com

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