10.3: Therapeutic Biotechnology

Biotech Focus

The discovery of insulin in 1922, by Frederick Banting and Charles Best, marked a major breakthrough in medicine and therapy for patients with Type I diabetes. However until the 1970s, Type I diabetics had to rely upon insulin isolated from the pancreas of either cattle or pigs. This necessitated the slaughter of hundreds of thousands of animals each year and could also produce significant immune reactions in certain diabetics. In response, in 1978, David Goeddel and his colleagues at Genentech, successfully inserted the gene for human insulin into E.coli cells and confirmed its production by these genetically-modified bacteria. In 1982, recombinant human insulin was approved by the FDA and introduced to the market as Humulin® - the first genetically-engineered drug to be approved for human use.

Introduction

Therapeutic biotechnology uses biological systems, organisms, or processes for the development of medical treatments that diagnose, prevent, or cure diseases and disorders. This area of biotechnology relies on the use of tools like recombinant DNA (rDNA) technology, genetic engineering, and gene therapy to create targeted therapies for conditions such as cancer, genetic disorders, autoimmune diseases, and infections. Using these tools, researchers have developed specialized therapies, including monoclonal antibodies, recombinant proteins, and vaccines. Through therapeutic biotechnology, researchers have transformed medicine and significantly improved human health.

Learning Objectives

Through therapeutic biotechnology, researchers can create medical treatments that diagnose, prevent, or cure diseases and disorders. At the end of this page, you will be able to:

• Define therapeutic biotechnology
• Explain gene therapy and some of it approaches
• List some of the pros and cons of viral vectors used in gene therapy
• Explain CAR-T gene therapy and how it can be used to cure cancer
• Explain the potential role of CRISPR in gene therapy
• Define what a therapeutic protein is and explain some major examples
• Explain the difference between a traditional and therapeutic vaccine
• Explain how an mRNA vaccine is made

Gene Therapy

Gene therapy is a therapeutic technique that aims to correct an underlying genetic problem by modifying a person’s genes. Through gene therapy a disease can be cured rather than treated. The first step of gene therapy is to identify the faulty gene. Often, the gene at the heart of the issue has been mutated in some way, preventing it from being made or functioning properly. The mutated gene (or genes) can be identified through DNA sequencing and genomic analysis. Once the gene (or genes) has been identified, researchers can begin to design a therapeutic strategy using a gene therapy approach.

There are several approaches used in gene therapy, including:

• Replacing the faulty or missing gene with a normal copy (i.e., "traditional" gene therapy)
• Inactivating or “silencing” the malfunctioning gene using miRNA or siRNA technologies
• Introduction of a new or modified gene to counteract the disease or improve cell function
• Correction of the gene mutation in situ through gene editing like CRISPR technology

Traditional gene therapy involves the replacement of a faulty or missing gene through the introduction of "normal" one. This approach has been used in the treatment of diseases, like cystic fibrosis. Cystic fibrosis can be caused by one of several mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene, a gene that codes for a chloride channel found in the plasma membrane of epithelial cells. This chloride channel regulates the movement of chloride and sodium ions across the plasma membrane and controls the balance of salt and water across the surfaces of epithelial tissues lining the respiratory and digestive systems. When this channel functions properly, it helps keep the mucus coating these epithelial linings thin and slippery. Mutations in the CFTR gene lead to a faulty or absent CFTR protein, resulting in thick and sticky mucus that clogs airways, traps bacteria, and blocks pancreatic enzymes (Figure \(\PageIndex{1}\)). Patients suffering from cystic fibrosis have significant breathing problems, lung infections, and digestive issues. Most cystic fibrosis patients will die between the ages of 35 and 50. The introduction of a functional CFTR gene through gene therapy could potentially cure this disease.

Figure \(\PageIndex{1}\): Cystic fibrosis (CF) and gene therapy. CF is caused by an abnormal CFTR channel protein. The CFTR controls the flow of sodium and chloride ions in and out of the epithelial cells lining the respiratory and digestive systems. In normal CFTR channel function (left panel), the channel is open and chloride ions (Cl-) and sodium ions (Na+) diffuse down their concentration gradients (black arrows). As a result, water (H₂0) moves out of the cell through osmosis, minimizing the thickness of the mucus layer covering the cell. In abnormal CFTR function (right panel), the channel is closed to ion diffusion. Abnormal ion diffusion prevents the osmotic movement of water out of the cell and a thick build-up of mucus outside of the cell results. (CFTR Function in CF by Patricia Zuk, CC BY 4.0; adapted from CFTR Protein Panels by Lbudd14, CC BY-SA 3.0)

Traditional gene therapy to treat diseases like cystic fibrosis was first introduced over thirty years ago to limited success. Challenges associated with the limitations in the size of genes that could be corrected, coupled to restrictions in the diseases that could be targeted, prompted researchers to explore other approaches. One growing research is the use of gene therapy to deliver microRNA (miRNA) or small interfering RNA (siRNA). As described in Chapter 5.5:Functional Genomics, miRNAs are small non-coding RNA molecules (about 22 nucleotides long) that regulate gene expression by binding to messenger RNAs (mRNAs) and either blocking their translation or causing their degradation. In gene therapy, therapeutic miRNAs could be designed and delivered to down-regulate disease-causing genes, such as oncogenes in cancer. In some diseases, certain miRNAs are underexpressed. By replacing them, normal gene regulation and cell function could be restored. Finally, diseases caused by overactive miRNAs that silence beneficial genes could be inhibited by gene therapy-mediated delivery of anti-miRs or "miRNA sponges", a synthetic or naturally occurring RNA molecule designed to bind (i.e."soak up") microRNAs.

No matter the approach, there are some common steps used in gene therapy. The therapeutic gene is inserted into a delivery system which is usually a viral vector that has been engineered to be safe and non-replicating. The genetic material is then introduced either directly into a patient through intravenous or intramuscular injection (i.e., in vivo introduction), or a patient's cells can be removed for genetic manipulation and then reintroduced (i.e., ex vivo introduction). Once inside the cells, the new gene will be read by the cell machinery, allowing it to produce a functional protein that restores normal function or fights the disease.

While alternatives to the use of viral vectors are being explored in gene therapy, including the packaging of the corrective genetic material into lipid nanoparticles or direct electroporation into cells, the most common delivery system still remains the viral vector. The choice of viral vector will depend on the size of the gene being cloned, the targeted cell, and the possible immune reaction of the patient.

Common viral vectors include:

1. Adeno-Associated Virus (AAV)
   • Pros: Low immune response, doesn't integrate into the genome and reduces risk of genetic mutations, long-lasting expression in non-dividing cells
   • Cons: Small gene capacity (~4.7 kb), requires repeated dosing in dividing cells
   • Currently used for: Eye disorders (e.g., Luxturna for retinal disease), spinal muscular atrophy
2. Adenovirus
   • Pros: High gene capacity, efficient delivery to many cell types, fast expression
   • Cons: Strong immune response, usually doesn't integrate into DNA (i.e., temporary expression)
   • Currently used for: Cancer gene therapy, vaccines (e.g., some COVID-19 vaccines)
3. Lentivirus
   • Pros: Integrates into the genome for long-term expression, works well in dividing and non-dividing cells
   • Cons: Risk of insertional mutagenesis due to genomic integration, more complex production
   • Currently used for: Blood disorders (e.g., sickle cell disease, β-thalassemia), HIV-related therapies

CAR-T Gene Therapy

Ex vivo gene therapy methods (also called cell-based gene therapy) involve the modification of a patient's cells outside of their body, followed by their re-introduction. The most successful method being used currently is CAR-T gene therapy for the treatment of a variety of blood cancers, including leukemia, lymphoma, and multiple myeloma. In CAR-T therapy, a patient's T cells are extracted from their blood and genetically modified in a lab through the introduction of a gene for the chimeric antigen receptor/CAR. Expression of this gene results in the expression of the CAR on the surface of the T cell. The modified T cells are expanded in a cell culture facility and infused into the patient's bloodstream (Figure \(\PageIndex{2}\)). Through the CAR, the T cells can bind antigens specific to cancer cells and destroy them. While showing promise, significant side effects of CAR-T therapy have been observed including neurological issues and "cytokine release syndrome" which results in fever, shortness of breath, low blood pressure, and in rare cases, heart or lung issues. The FDA is current investigating instances of secondary T-cell lymphomas in people who have been treated with CAR T-cell therapy.

Figure \(\PageIndex{2}\): CAR-T gene therapy. T cells are isolated from a patient and genetically modified by introducing the gene for the Chimeric Antigen Receptor (CAR gene). Gene expression of this gene leads to the expression of the CAR on the surface of T cells. The modified T cells are expanded in number and then infused back into the patient for the targeted of cancerous cells. (CAR-T Therapy Overview by Patricia Zuk, CC BY 4.0)

Gene Therapy and CRISPR

Early trials in gene therapy approaches showed promise but demonstrated limited effectiveness due to challenges like immune responses and short-lived expression. Newer approaches include CRISPR-based gene editing, which aims to correct the mutation directly in the patient’s DNA without the need to introduce additional genetic information. As outlined in Chapter 8.1: Applications of Animal Biotechnology, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is derived from a bacterial defense mechanism that protects bacteria from the integration of viral DNA into the bacterial genome. CRISPR uses a guide RNA and the Cas9 enzyme to precisely cut DNA at specific locations in the genome. Researchers are exploring its use in correcting, deleting, or replacing genes, making it a powerful tool in combination with gene therapy.

Current gene therapy applications using CRISPR include:

• Sickle Cell Disease: used ex vivo to edit a patient's blood stem cells in order to "reactivate" fetal hemoglobin and compensate for the defective hemoglobin gene. The edited cells are reinfused into the patient.
• Leber Congenital Amaurosis (LCA): uses CRISPR to edit retinal cells in vivo to restore vision in patients with this inherited form of blindness
• Beta-Thalassemia: similar to sickle cell, CRISPR is being used to edit the genome of bone marrow stem cells to boost healthy hemoglobin production

While the results are exciting, there are limitations to gene therapy and CRISPR. Chief among them is a concern about "off-target" effects. CRISPR may cut unintended parts of the genome and could introduce unintended mutations. Delivery challenges also exist. Getting CRISPR components safely and efficiently into the right cells using viral vectors or nanoparticles is still an obstacle. Finally, an immune response to Cas9 may result in a significant decline in the effectiveness of this technology.

Concept in Action

CRISPR Gene Editing of Sickle-Cell Anemia

Therapeutic Proteins

Therapeutic biotechnology is critical for the development of therapeutic proteins. A therapeutic protein is a biologically-engineered protein that is used to treat disease by replacing, enhancing, or modifying a specific physiological function or functions. Therapeutic proteins are typically produced by genetically-modified bacteria, yeast, or mammalian cells that have been modified using rDNA technology. However, therapeutic proteins can also be produced by genetically-modified organisms (i.e., transgenic organisms), such as plants and animals.

There are numerous types of therapeutic proteins with specific functions, including:

• Enzyme Replacement Therapy (ERT) proteins: replace missing or defective enzymes in patients with genetic disorders; examples include:
  • lactase: produced to treat lactose intolerance
  • alpha-glucosidase (i.e., Myozyme^TM); used to treat for Pompe disease
• Hormones: used to regulate biological processes like metabolism, growth, and reproduction; examples include:
  • Insulin: produced by either genetically-modified bacteria or yeast; used to treat Type I diabetes without the need for animal donors
  • Human Growth Hormone (HGH): produced using genetically-engineered bacteria; used to treat growth hormone deficiencies in children and adults.
  • Erythropoietin (EPO): produced by genetically-modified mammalian cells; used to stimulate red blood cell production in patients with anemia; also used/abused by endurance athletes such as marathon runners, triathletes, and long-distance cyclers
• Clotting Factors: used in the treatment of blood clotting disorders; examples include:
  • Factor VIII and IX: produced using genetically-engineered mammalian cells; used to treat hemophilia and ensure safety and supply without relying on human donors.
• Monoclonal Antibodies: target specific antigens for treating cancer, autoimmune diseases, and infections; examples include:
  • Trastuzumab^TM (anti-HER2 receptor antibody; Herceptin): produced by genetically-modified mammalian cells; used to treat certain types of breast and gastric cancers by binding the Human Epidermal Growth Factor Receptor Type 2 (HER2), blocking its function and triggering its destruction by the immune system
  • Adalimumab^TM (anti-TNF alpha antibody; Humira): produced by genetically-modified mammalian cells; used to treat certain autoimmune disorders by blocking the function of Tumor Necrosis Factor-alpha (TNF-alpha)
• Immune modulators: produced to stimulate or regulate the immune system; examples include
  • Interferons: produced by genetically-modified bacteria or yeast; used to treat certain cancers, multiple sclerosis, and viral infections
  • Interleukin-2: produced by genetically-modified bacteria; used to boost the immune response during chemotherapy

Most therapeutic proteins are produced by genetically-modified bacteria, yeast, or mammalian cells. The production of the protein starts with the selection of the desired gene. This gene is then cloned into a suitable plasmid and the resulting recombinant plasmid is then introduced into the genome of the "host cell" (e.g bacteria or mammalian cell). The modified cells are then grown in the laboratory and the protein isolated. Following extensive confirmation that the therapeutic protein is made properly and functions effectively, the protein is then introduced into the commercial market and made through precise manufacturing processes. To learn more about this DNA cloning process, go to Chapter 4.2: Creating Recombinant DNA.

While the approach for genetically-modifying cells for the production of therapeutic proteins is generally the same, the technique used to introduce the genetic information into a cell depends on the cell type (Figure \(\PageIndex{3}\)). For bacteria, this technique is called transformation. For mammalian cells, genetic information is introduced through liposomal transfection. In transformation, bacteria are treated with ice-cold solutions to weaken their outer wall and plasma membrane, making them "competent" for transformation. They are then mixed with the recombinant plasmid and "heat shocked" to induce internalization of the plasmid. For transfection, the recombinant plasmid is coupled to microscopic liposomes that bind to the mammalian cells and are internalized through endocytosis. To learn about transformation, go to Chapter 13.6 Lab Technique: Bacterial Transformation. To learn about transfection, go to Chapter 13.10 Lab Technique: Liposomal Transfection into Mammalian Cells.

Figure \(\PageIndex{3}\): Transformation vs. Transfection. In transformation, competent bacterial cells undergo a "heat shock" to internalize DNA such as plasmid DNA. The transformed bacteria with its incorporated plasmid DNA begin expression of the new gene product. In transfection, DNA (e.g. plasmid DNA) is added to liposomes to form a liposome/DNA complex. The complex binds eukaryotic cells and is internalized into the cell through endocytosis. Once inside the cell, the complex undergoes uncoupling and the DNA is released into the cytoplasm. (Transformation vs Transfection by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2024))

Vaccines

There are two basic kinds of vaccines: traditional and therapeutic. The traditional vaccine stimulates the immune system in order to prevent an infection, whereas a therapeutic vaccine is designed to treat cancer, an existing disease, or chronic infection (like HIV or hepatitis) rather than prevent it. rDNA technology is used in the creation of both types of vaccines. To produce a vaccine using rDNA technology, highly specific viral or bacterial proteins that trigger an immune response without causing disease are produced in the lab. The process for this is very similar to recombinant protein production. A gene crucial for the pathogen’s ability to infect a host cell or illicit an immune response is cloned into a plasmid and introduced into a host cell for expression. The expressed protein is purified extensively to ensure that the vaccine contains only the desired protein and no harmful components from the host organism. The purified protein is then used to formulate a vaccine.

While the typical vaccine is made using expressed proteins, the process can be time-consuming. However, the development of mRNA vaccines has enabled scientists to dramatically cut down the generation time for a vaccine. Making an mRNA vaccine uses a few steps in common with the traditional vaccine described above (Figure \(\PageIndex{4}\)). A gene for a target protein is identified and cloned into a plasmid. However, rather than expressing this gene as a protein in a cell, the mRNA transcript is produced by a cell-free system and isolated. The mRNA is then coupled to lipid nanoparticles that protect the delicate mRNA. In the final step, the mRNA-nanoparticle mixture is injected into muscle tissue whereupon the mRNA enters into the muscle cells and is expressed as a protein. The expressed protein is displayed on the surface of the muscle cell and triggers a immune reaction.

Figure \(\PageIndex{4}\): mRNA vaccine production. Vaccines against the spike protein of the Sars-CoV-2 virus can be made using mRNA. mRNA that codes for the spike protein is synthesized using a cell-free system (mRNA production). The mRNA particles are encapsulated into lipid nanoparticles (lipid nanoparticle encapsulation of mRNA). The mRNA-lipid nanoparticles are used to vaccinate host cells (vaccination of cells with mRNA vaccine). Once inside the cell, the mRNA is released from the nanoparticle and translated by the host cell's ribosomes. The resulting spike proteins are embedded within the cell membrane and recognized by the host's immune cells. Antibodies to the spike protein are made by the host (antibody production by immune system). (mRNA vaccine production by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2024))

Molecular Protocols

• Lab Technique: Bacterial Transformation
• Lab Technique: Transfection of Mammalian Cells

Key Concepts

Therapeutic biotechnology can be used to treat a wide range of diseases and disorders. Some major concepts to remember are:

• therapeutic biotechnology uses biotechnology tools to diagnose, treat, cure, or prevent diseases or disorders
• gene therapy can potentially treat or cure diseases through the introduction of a gene (or genes) that either replaces a "faulty" one or counteracts its harmful effects
• genes can be introduced for gene therapy using viral vectors like adenovirus, adeno-associated virus, or lentivirus
• new methods for the introduction of genes for gene therapy include the use of nanoparticles
• gene therapy can either be performed by introducing corrective genetic information into an organism directly (in vivo approach) or by removing the affected cells, genetically manipulating them, and then re-introducing them back into the patient (ex vivo approach)
• CAR-T therapy is a type of gene therapy that genetically-manipulates a patients T cells in order to treat cancer
• CRISPR technologies are currently being explored for use in gene therapy
• most therapeutic proteins are made using bacteria, yeast, or mammalian cells that have been modified with recombinant DNA
• some therapeutic proteins are made through genetically-modifying the entire organism with recombinant DNA
• examples of therapeutic proteins are monoclonal antibodies, hormones, an immune modulators
• therapeutic vaccines use recombinant DNA to produce specific antigens for vaccine production

Glossary

CAR (Chimeric Antigen Receptor) - a synthetic receptor engineered into immune cells (usually T cells) that allows them to recognize and bind to specific proteins on the surface of cancer cells

CAR-T therapy (Chimeric Antigen Receptor T-cell Therapy) - a type of immunotherapy where a patient's T cells are collected, genetically modified in the lab to express CARs, and then re-infused for targeted destruction of a cancer cell

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) - a gene-editing tool that uses a guide RNA and a Cas9 enzyme to find and cut specific DNA sequences

Ex vivo - refers to a process or event that happens outside of the body

Gene therapy - a technique that treats or prevents diseases by modifying the genetic make-up of a person using viral or non-viral vectors

In vivo - refers to a process or event that happens inside of a body

Liposome - a spherical vesicle made of phospholipids that mimics the structure of a cell membrane; used in transfection of mammalian cells

mRNA (messenger RNA) - a sequence of RNA produced through the process of transcription; specifies the sequence of amino acids following translation; comprised of a coding sequence and flanked by a 5' methylated cap and a 3' poly-A tail

mRNA vaccine - a vaccine containing mRNA molecules surrounded by lipid nanoparticles

Nanoparticle - a tiny particle that ranges in size from 1 to 100 nanometers (nm) in at least one dimension; can be made of materials such as biodegradable polymers, metals, and lipids

Recombinant DNA (rDNA) - DNA that is created in a laboratory by combining genetic material from different sources

Therapeutic protein - a protein-based compound used to treat, prevent, or cure diseases and medical conditions; often produced using recombinant DNA technologies

Therapeutic vaccine - a vaccine used to prevent diseases; often produced using recombinant DNA technologies

Transformation - the process of inserting DNA into a bacterial host cell

Transfection - the process of introducing DNA into a mammalian cell using liposomes

Vaccine - a biological preparation that stimulates the immune system to protect against a specific disease