9. Medical Biotech & Gene Therapy

What is Medical Biotechnology?

• Medical biotechnology can be defined as a branch of biotech that uses specialised techniques to manipulate microorganisms, plants, animals, and human tissues with the overall aim of understanding, treating and, if possible, curing human disease

• Medical biotech builds on our basic understanding of cell bio, genetics, molecular bio, biochem, microbiology, physiology, pharmacology and immunology

Why is medical biotech important?

• prolong life

• ease patients suffering

• increase accessibility for patients with disabilities

• AMR - age standard mortality rate - has been decreasing due to biotech advancements

What are some of the historical moments in medical biotech?

• 1797 - Jenner immunises humans with cowpox virus to protect against smallpox

• 1919 - first use of term biotechnology in print

• 1928 - Fleming discovered penicillin

• 1938 - term molecular bio coined

What is a stop codon in genetic coding?

• a stop codon will not translate to one of the 22 amino acids

• stop codons are: UAA, UAG, UGA

What is Transcription?

• Initiation - RNA polymerase binds to promoter region of DNA which signals DNA to unwind

• Elongation - is the addition of nucleotides to mRNA strand. RNA polymerase reads unwound DNA strand and builds mRNA molecule using complimentary base pairs: A binds to U in RNA

• Termination - end of transcription. Occurs when RNA polymerase crosses a stop/termination sequence in the gene. mRNA strand is complete and detaches from DNA

What are the steps for translation?

• Protein synthesis process.

• In E. Coli process uses: ribosome, mRNA template, initiation factors and initiator tRNA

• Initiation: initiator tRNA interacts with the start codon AUG. GTP is translation energy source. Once AUG is identified, 50S subunit binds to the complex of met-tRNAi, mRNA and the 30S subunit.

• Elongation: mRNA template provides specificity and as the ribosome moves along mRNA, each mRNA codon coms into register and specific binding with corresponding charged tRNA anticodon is ensured. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Charged tRNAs enter A site and shift to the P site then E site with each single codon ‘step’ of ribosome. Peptide bonds form between amino acid attached to P site tRNA. amino acid bound to P site tRNA is also linked to growing polypeptide chain. As ribosome steps across mRNA, former P site tRNA enters the E site and detaches from amino acid and is expelled.

• Termination: occurs when a nonsense codon is encountered (UAA, AUG or UGA). Upon aligning with A site, these nonsense codons are recognised by release factors that instruct peptidyl transferase to add a water molecule to carboxyl end of P site amino acid. This reaction forces P site amino acid to detach from tRNA and the newly made protein is released. ribosomal subunits dissociate from mRNA and each other to be recruited into a different translation initiation complex.

What are the steps to make monoclonal antibodies?

1. Human antibody genes are put into a mouse

2. Mouse is infected causing it to make human antibody producing cells (B-cells)

3. These cells are removed from mouse and fused with a tumour cell

4. Now we have a tumour cell tht is constantly producing antibodies and more cells like itself

What are the applications of monoclonal antibodies?

• Diagnostic tools

• Locating environmental pollutants

• Detect harmful microorganisms in food

• Detect pathogens of infectious diseases

• Detect specific antigens and to localise cells or specific proteins

• Therapeutic monoclonal antibodies - e.g. Avastin - monoclonal antibody that can inhibit angiogenesis by blocking vascular endothelial growth factor (VEGF)

What is gene therapy?

• the use of DNA as a pharmaceutical agent to treat diseases

• Derives from the idea that DNA can be used to supplement or alter genes within an individual cell as a therapy to treat disease

• Most common form involves using DNA that encodes a functional therapeutic gene to replace a mutated gene

• Two types: somatic and germ line gene therapy

What is bubble boy disease known as scientifically? What was done to treat it?

• Severe combined immunodeficiency disease (SCID)

• victims are vulnerable to infectious diseases and have to live in a sterile environment

• Treatments: Bone marrow transplantation, enzyme replacement therapy, Gene therapy

• Gene therapy has 2 ways:

1. X-SCID - IL2R gene located on X-chromosome, IL2R allows communication of T and B cells

2. ADA-SCID - adenosine deaminase encoding gene on chromosome 20 - without adenosine deaminase our body cannot break down deoxyadenosine which is toxic and destroys B and T cells.

What is the process for ADA-SCID gene therapy?

1. haematopoietic stem cells are isolated

2. Reprogrammed to contain correct gene - use a retrovirus to delvier the gene

3. Reprogrammed haematopoietic stem cells are then transplanted back into the patient

What are the gene therapy carriers/delivery methods?

• VECTOR - the carrier molecule use to deliver therapeutic gene to patients target gene

1. Retrovirus: one that will randomly integrate into the genome

2. Adenovirus, will not integrate into the genome

3. Liposome: protective lipid bilayer where micelles facilitate DNA transfer

4. Gene gun

What are features of the liposome vector?

• micelle facilitates DNA transfer

• DNA requires cationic liposomes embedded with cholesterol

• Liposome must protect DNA from nucleases, mediate rapid internalisation and be non toxic

Where do embryonic stem cells come from?

mass in the blastocyst

What are the key properties of stem cells?

• self -renewal/copying - maintaining the stem cell pool

• Pluripotency - specialisation and differentiation to replace dead or damaged cells throughout life

What are the types of stem cells?

• Embryonic stem cells (ESCs) - from the inner cell mass of blastocyst, lab culture allows for growth of more cells, pluripotent and can differentiate into anything apart from placenta cells

• Adult Stem Cells (Adult SCs) - multipotent, can only differentiate into a few cells types, specialised for certain tissue areas/organs, from bone marrow or circulation/resident tissue

• Induced Pluripotent stem cells (iPSCs) - genetic reprogramming of somatic cells, take a cell from the body and induce pluripotent stem cell to make it behave like an embryonic stem cell that can be multiplied in lab cultures and differentiated into any types of specialised cells - no need for embryos

What types of treatment can be given to patients?

• surgery

• molecules/drugs

• antibodies

• Genes

• Stem cells

What is one new therapy method for Heart diseases?

• Adult stem cell therapy

• cells taken from bone marrow can create endothelial cells and angiogenesis and vasculogenesis can improve heart function

• Cells from adipose tissue form muscle stem cells and can help heart function

• Cardiac tissue can form cardiac progenitor cells or CDC to form cardiomyocite and cardiogenesis to improve heart function

How does iPSCs and genome editing cell therapies work?

• iPSCs cell therapy: reprogramming cells from patient to form iPSCs and if it is a genetic disease, they undergo gene editing to create healthier ‘corrected’ iPSCs and are then differentiated and used in cell therapies or if not genetic, the cells just undergo differentiation before being used

How are genes edited using CRISPR?

• CRISPR/Cas9

• Guide RNA sticks to Cas9 and directs Cas9 to the target DNA, Guide RNA contains sequence that matches target DNA

• When target identified, Cas9 unzips the target DNA and the guide RNA matches up to it Once matched up, Cas9 uses molecular scissors to cut the target DNA

• Once cut, DNA can be disabled or altered

What is the traditional drug development timeline and the treatment timeline?

1. Devlopment: selection of therapeutic molecule → pre-clinical testing on transgenic cell lines/small animals/large animals → slow and costly build up to clinical testing

2. Patient treatment: Diseased patient identified → possible therapies investigated through patient history, clinical exams, testing of multiple drugs → slow selection of the best drug

what is the precision medicine initiative?

• an enterprise to revolutionise medicine and generate scientific evidence needed to move the concept of precision medicine into everyday clinical practice

• built on principle that that treatment and prevention needs to take into account individual variability in genes, environment, and lifestyle of each person.

How were stem cells affected by precision medicine?

• iPSC saw a paradigm shift in stem cell biology and translation

• they can be made for each individual based on their own somatic cells being turned into stem cells and engineered for each person.

What were the results/relevance of patient-specific iPSCs as a model for familial dilated cardiomyopathy?

• iPSC-CMs generated from a 7-member family with DCM helped to pinpoint the DNA sequence point mutation. diseased DCM iPSC-CMs showed altered regulation of calcium ions, decreased contractility and abnormal distribution of sarcomeric alpha-actinin

• Treatment with beta-adrenergic agent causes increases cellular stress and tretment with beta blocker improved function of DCM iPSC-CMs

What are genetic vs Acquired vs Multifactorial causes of heart disease?

• Genetic (primary cardiomyopathy) : HCM, DCM, ion channel disorders etc

• Acquired (primary cardiomyopathy): inflammatory myocarditis, stress-provoked, DCM

• Secondary cardiomyopathies can be cuased through: toxicity to drugs/radiation/heavy metals, endocrine disruption, nutritional deficiency, infiltrative etc

• or there are other cardiovascular abnormalities such as coronary artery disease, congenital HD, etc

• All of these lead to cardiac remodelling and heart failure

What is the envisioned iPSC based precision method development timeline?

1. iPSC-based drug discover: diseased patient used for target selection and in vitro screening done to produce patient-specific iPSCs

2. Personalised therapy and testing: disease specific target cells made and in vitro screening for therapeutics done, drug tested on animal models and then goes through human clinical trials

• once FDA approved the target specific cells can be repurposed

• this has a reduced cost and will have toxicity tests

What is the iPSCS for clinical trial in a dish process?

• collect somatic tissue from patient and establish somatic cell line in dish

• store patient sample and introduce reprogramming conscription factors to form iPSC

• Expand iPSC cell culture and then undergo genetic modification and characterisation for in vitro differentiation to any desired cell type

• Assay cells made in disease and safety models

• Clinical trialling