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Bioengineering

 

 

A rapidly increasing area of immunotherapy are new methods using immunogenetic and immunogenomic (you may want to do a quick refresher on those sciences in Blog #10) methods. They include:

  • “editing” cells and genes;

  • replacing cells and genes;

  • transplantation of cells and genes including direct cell transplants and using specialized cells called stem cells in a process called “regenerative medicine:;

  • gene replacement procedures including something called CAR-T or CAAR-T cell therapies which we’ll be explaining in some depth in Blog #25; and

  • gene editing procedures including CRISPR-Cas9 (and 13) therapies, again to be explained in Blog #26.

 

All of these immunotherapies are targeted, organ-specific treatments, as in stem cell therapies, as well as disseminated therapies treating the patient’s whole genome, as in CAR-T, CAAR-T and CRISPR therapies. Some of these treatment techniques have similar applications but with different treatment goals as in autoimmune diseases, genetic disorders, cancers, and numerous other congenital, acquired, and chronic conditions. These innovative and “disruptive” biomedical and cellular therapies enjoy piggybacking off the successes of other genetic and cancer treatments and vice versa. The impact of these procedures and, granted their complexities, are of such consequence to immunotherapy that each deserves its own blog. So let’s start with stem cell therapies and then address each subsequent procedure in detail in separate blogs.

 

Blog #22: Immunotherapeutic procedures (PART 1: Stem cell transplantation therapy)

Stem cells are cells within the body originate during embryologic development and are called embryonic stem (ES) cells). During early life and growth these cells are non-specific or “undifferentiated” and have the potential to develop into many, and any different types of future adult stem cells found in organs and tissues in the body. They also differentiate into red blood cells and white blood cells (WBCs) which you will remember include lymphocytes or immune cells (Figure 5.2). Adult stem cells serve as a repair system for the body. In some organs, such as the gut and bone marrow, they regularly divide to repair and replace worn-out or damaged tissues. When these cells, particularly the more undifferentiated types, are used in cell-based therapies to treat disease, the process is referred to as regenerative or reparative medicine.

 

The clinical value of the ES cells lies in their ability to form any kind of cell, a process called “differentiation.” This means they can start as embryonic, or their more scientific name, pluripotent stem cells (PSCs) and differentiate into specific adult stem cells and essentially repair, replace and regenerate normal healthy tissue. In a more sinister role, they can also differentiate into disease-oriented progenitor cells. But, perhaps the most important potential application of human stem cells is their generation of cells and tissues that could be used for cell-based therapies like “stem-cell transplantation.” Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including macular degeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis (RA).

 

Stem cells can be readily harvested from bone marrow (called bone marrow transplant); adipose (fat) tissue, a bountiful source of stem cells; and other bodily tissues. They are then bioengineered or “induced” into undifferentiated pluripotent cells (iPSCs) suitable for transplantation into diseased and degenerated organs and body structures (e.g., diabetes, osteoarthritis, etc.). These iPSC cells then regenerate and begin to replace the abnormal cells with new, normal cells and even potentially functioning organs, a process called organ morphogenesis (Figure 5.2). Currently, muscle and bone tissue are particularly amenable to cell and tissue regeneration.

 

Stem cell transplantation procedures include a number of methods to deliver targeted therapeutic genes into the body through direct delivery and/or cell delivery (Fig. 5.3). Direct delivery packages the gene into an engineered vehicle, like an attenuated (neutralized) virus that is injected into the patient, whereupon it penetrates the genome and the therapeutic gene is thus delivered to the targeted organ system. The weakness of this method of delivery includes the random integration of the gene into every chromosome in the patient’s body with unknown potential adverse effects. Conversely, the cell delivery method removes embryonic stem (ES) cells from the patient and introduce the “packaged gene” directly into those removed ES cells and then return back into the patient. The use of undifferentiated ES cells as the vehicle for gene retransplant to the patient offers additional specificity to the process where the ES cells can replicate only in the target organ.

 

The objective of stem cell transplantation therapy in immunology is to destroy the mature, long-lived, weakened immune cells and generate a new, properly functioning immune system. This process has enormous potential in autoimmune diseases, cancers, and other hereditary and acquired genetic mutations resulting in immune system compromise. The patient’s stem cells are used in a procedure known as autologous (from “one’s self”) stem cell transplantation. First, patients receive injections of a growth factor, that coaxes large numbers of undifferentiated stem cells to be released from the bone marrow into the bloodstream. These cells are then harvested from the blood, purified away from the body's mature immune cells, and stored. After sufficient quantities of these undifferentiated cells are obtained, the patient undergoes a regimen of cytotoxic, cell-killing drugs and/or radiation therapy that eliminates the body's abnormal mature immune cells. Then, the undifferentiated stem cells are returned to the patient via a blood transfusion into the circulation where they migrate to the bone marrow and begin to differentiate to become mature immune cells. The body’s immune system is then restored.

Blog #23:     Immunotherapeutic procedures (Part 2: Homograph transplantation)

 

A branch of immunotherapies of profound importance is that of organ, tissue, and cell transplantation. So too are the profound immunological challenges presented in such therapies. All of the considerations of innate and adaptive immunity come into play with homograph, aka allograph transplantation, that is transplantation from one donor to another of the same species (humans for this discussion) but with different genetic (genotype) makeup. Between the basic immunologic tenet of “self-versus-non-self” to the memory response of the immune system, success with homographic transplantation needs intricate blood typing for cellular receptor identification, and genetic matching of donor and recipient.

 

Organ failure (heart, liver, kidneys, etc.) is not unusual in humans, no less tissue injury and destruction by dermatological diseases (autoimmune and otherwise, e.g., accidents, and particularly, burns). So, the need to reduce or suppress the immune system’s natural reaction to a “foreign transplant” was an obvious necessity in homographic transplantation. Immunosuppressive drugs, from steroids to the strongest immunotherapeutic agents, proved capable of doing the job of controlling the immune response. The introduction of the immunosuppressant drug cyclosporine in 1983 revolutionized transplant medicine. But needless to say, these drugs would also reduce the patient’s fundamental (T cell, B cell, etc.) immune defenses against other non-self-invader, particularly opportunistic infectious agents.

 

A group of autoantigens (i.e., self) called isoantigens (blood antigens or HLA complex, all slightly different in individuals) are present in some members of the human species (subset) and not others. Blood transfusion or organ or tissue transplantation of isoantigens into a donor without isoantigens will produce an immune antibody response and result in a severe reaction or graft rejection. So, it is critical that donor blood types and isoantigens are properly matched with recipients.

 

The discovery of isoantigens led to a more dynamic form of transplantation, namely bone marrow transplant. Given that the bone marrow is a principal site of stem cell, blood cell, and immune cell development, its value in providing a “new” immune system to a qualified recipient with an isoantigen match is of obvious value in treating  autoimmune diseases and cancers. Bone marrow transplants may use cells from your own body, called autologous transplant, or from a donor, called allogeneic transplant. In either case, with a proper donor, the stem cells will yield a new, hopefully revitalized and disease-free immune system. By combining these newly developed immunotherapeutic procedures, new treatment modalities including animal to man or “xenotransplantation,” may be viable alternatives to allogeneic transplantation. Recently, a pig heart genetically modified by CRISPR-Cas9 (discussed in Blog #26) was successfully implanted in a 57-year-old male who survived for 2 months. According to the attending surgeons, heart failure resulted from a number of factors. As of this writing, there are no surviving xenotransplant patients.

Blog #24: Immunotherapeutic procedures (Part 3: Genetic engineering/modifications)

 

The use of the word “cure” in medicine always needs delicate consideration. If you recall, the only times I have used the word in earlier blogs was where I was describing the value of “removing the cause” in immune-inflammatory disease. The only place I would consider medical care’s potential to achieve a “cure” in health care, especially for autoimmune diseases and cancers lies in the current and evolving genetic therapies we will now discuss. They include CAR-T and CAAR-T cell replacement therapies, and CRISPR-Cas9 gene editing. Sometimes referred to as “genetic engineering” or “genetic modification,” these procedures can be defined as the direct manipulation of the genome using molecular biology engineering techniques. These engineering techniques for modifying genes can be applied in two very different ways: somatic genetic modification or modifications of selective genes of the body; and germline genetic modification or modifications of the entire genome.

 

Somatic genetic modification adds, cuts, or changes the genes in some of the cells of an existing person, typically to alleviate a medical condition. A number of these gene therapy techniques are now FDA-approved for specific conditions. Germline genetic modification, quite different from “gene editing,” is used to “change” the genes in eggs, sperm, or early embryos. A number of these therapies are also FDA-approved, but, as of this writing, they are under intense scrutiny because of serious issues in that genetic engineering stands the risk of going well beyond the science and safety of the field. Bioethical questions also abound regarding potential uses and misuses of this germline bioscience as well as AI applications expanding its potential beyond therapeutic purposes. To wit, some controversial uses of genetic engineering include (but are not limited to):

  • Human genetic enhancement: The intentional modification of the human genome to “improve” individuals;

  • Human germline genome editing: Introducing heritable changes to sperm, eggs, or embryos;

  • Eugenics: (Eugenics is from a Greek word meaning “normal genes.”) Its modern definition describes it as the attempt to direct human heredity and evolution to ensure procreative advantage to more “desirable” human beings and to discourage or limit reproduction by the less desirable (that sounds like a pretty ugly proposition to me);

  • Genetic cloning: Cloning describes the processes used to create an exact genetic replica of another cell, tissue, or organism. The copied material that has the same genetic makeup as the original is referred to as a clone. Of course, the use of genetic cloning for monoclonal antibodies as discussed in Blog #21 is an enormously valuable procedure.

 

The ethics and pros and cons of all of these techniques are under excruciating analysis and review by international bioethics groups. Undoubtedly laws and regulations will be instituted in the coming years to mitigate the dangers of these technologies while maximizing their value in healthcare. As such, in the next few blogs we’ll look at each of the procedures and their therapeutic applications and implications.

Blog #25: Immunotherapeutic procedures (Part 4: CAR-T & CAAR-T cell therapy)

The word “chimeric” in biology means using two or more biological components linked to form or perform a new biological process. Chimeric antigen receptor T cells (CAR-T cells) are T cells that have been genetically engineered to give them the new ability to target a specific protein. The receptors are “chimeric” meaning they combine both antigen-binding and T-cell activating functions into a single receptor. The initial premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells and thus, more effectively target and destroy them (more on this to be discussed in Blog #27 on cancer). Similar to cancer treatment by targeting tumor-associated antigens expressed on the surface of tumor cells, CAR-T cells are now being modified to treat autoimmune diseases as well by targeting specific autoantigens or antibodies. This type of CAR-T cell immunotherapy is referred to as chimeric autoantibody receptor T or

CAAR-T cell and CAR-Treg which chimerically targets Treg cells [from Blog #5] to help regulate and suppress immune activity suspected of producing an autoimmune disease.

 

CAAR-T-cell immunotherapy (Figure 5.4) begins by removing a patient’s T lymphocytes and transducing or infusing them with a DNA molecule called a plasmid vector, distinct from the cell’s DNA and engineered to include a manipulated or cloned gene sequence or a patient’s stem cells that can be targeted for an autoantigen producing an autoimmune disease. These modifying T cells are then transferred back into the patient’s bloodstream through a single infusion (Fig. 5.4). The modified T lymphocytes begin to enhance the patient’s immune response by targeting the autoantigen protein. Known as autologous CAAR-T-cell therapy, this treatment is showing promising results in numerous autoimmune diseases. A similar process will be presented in Blog #32 on cancer treatment using encoded tumor antigens to elicit a targeted immune response to a cancer. Can you begin to see the potential of this elegant immunotherapeutic strategy.

Blog #26:     Immunotherapeutic procedures (Part 5: CRISPR-Cas9 therapy)  

One of the effective ways of treating autoimmune disease is to identify the “signature” of offending genes, that is the “gene expression” or the number of RNA molecules it is producing. In autoimmune and cancer expression of these offending genes are abnormal. The identification of these genes is accomplished by using a technique called “single-cell RNA sequencing” (scRNA-seq), or more specifically, TIDE (for Tumor Immune Dysfunction and Exclusion) for autoimmune genes. With this information, a revolutionary procedure called CRISPR-Cas9 (“Clustered regularly interspaced short palindromic repeats"). This is a family of DNA sequences found in the genomes of organisms where the DNA is in the cell cytoplasm rather than its nucleus – this is explained in a bit more understandable language ahead, so feel free to forget this last sentence). Cas9 is an enzyme sometimes referred to as “the scissor

protein.” In essence, the procedure is an RNA-guided genome editing technology being used to reengineer T cells.

 

Forgive me for getting too deep into the weeds on this technology, but it really is worth trying to understand its science. It already is a vital part of immunotherapy and will continue to expand dramatically in the coming years. So bear with me, reread it if necessary and you’ll benefit from understanding it.

 

Similar to the way bacteria defend against viral invasion, CRISPR-Cas9 is used to edit the genome by creating, identifying and targeting DNA breaks that will trigger specific DNA repair. When considering “next-generation” in genetic processing, the “central dogma of molecular biology” from Blog #10, the goal is to control the protein synthesis that produces all of our body’s tissues and cells. We can alter this process by altering the genome sequences using a method of editing the base compounds of genes. As these technologies continue to mature, it is becoming increasingly possible to efficiently and accurately alter cellular genomes.

 

The CRISPR-Cas9 system (Figure 5.5) creates a small piece of RNA (Cas9) that attaches or binds to a specific target sequence of DNA identified by NGS (next generation sequencing, from Blog #10) in a genome. The RNA also binds to the Cas9 enzyme and is used to recognize the DNA sequence. The Cas9 enzyme, acting like a “scissor” cuts the DNA at the targeted location. Once the DNA is cut, the cell’s DNA uses its repair machinery to add or delete pieces of genetic material, or it can make changes to the DNA by replacing an existing segment with a customized or “edited” DNA sequence. It was first thought that the stitching back together of the genetic material after the CRISPR-Cas9 procedure was random. But subsequent studies using AI to predict repairs made to DNA snipped with Cas9 confirmed that the edits aren’t random at all but rather follow the newly programmed genetic material.

 

It is worth noting here that in October 2020, the Nobel Prize in Chemistry was awarded to two molecular biologists, Emmanuelle Charpentier and Jennifer Doudna for the development of this revolutionary genome editing technique often referred to as “genetic scissors.” The unfortunate aspect of these immunotherapeutic procedures, and the CAR-T cell therapies as well, are their exorbitant costs. Notwithstanding the significant benefits these therapies provide, the costs of FDA-approved CAR-T cell therapy and the CRISPR-Cas9 procedure range from $373,000 to $875,000 for a single treatment. Also, depending on the type of stem cell, regenerative procedures, prices can range from $5000 to $25,000 per procedure. Gene therapies are subject not only to the regulatory structure of the FDA, but also to the Office of Biotechnology Activities, and the Recombinant DNA Advisory Committee. Excessive regulatory oversight creates an elongated and expensive route to approval. By one estimate, approval for a gene therapy costs nearly $5 billion, that’s five times as much as the average cost of FDA drug approvals. Some insurers are beginning to provide partial coverage of FDA-approved gene therapies, but experimental treatments receive no third-party coverage other than limited humanitarian exemptions. Hopefully, as with other major therapeutic discoveries, the costs of providing these technologies will reduce over time.

 

Finally, a new CRISPR-Cas13 mRNA screen has been developed to establish guide RNAs for the COVID-19 coronavirus and human RNA segments that could be used in vaccines, therapeutics,  and diagnostics. We’ll defer a full discussion on that technology to Blog # 36 on infectious diseases, pandemics, and of course, COVID-19.

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