🦠Virology Unit 18 – Viruses as Tools in Biotech & Gene Therapy

Viral Basics and Structure

• Viruses are non-living infectious agents that require host cells to replicate
• Consist of genetic material (DNA or RNA) encapsulated by a protein coat called a capsid
• May have an additional lipid envelope surrounding the capsid (enveloped viruses)
• Capsid proteins determine the shape of the virus (icosahedral, helical, or complex)
• Viral genome can be single-stranded or double-stranded, linear or circular
• Size of viral genomes varies widely, ranging from a few thousand to millions of base pairs
• Lack cellular structures and metabolic processes, relying entirely on host cell machinery for replication
• Bind to specific receptors on host cell surface to initiate infection

Genetic Manipulation of Viruses

• Viruses can be genetically engineered to express foreign genes or modify their properties
• Recombinant DNA technology allows the insertion, deletion, or modification of viral genes
• Viral genomes can be cloned into bacterial plasmids or yeast artificial chromosomes for manipulation
• Specific viral genes can be replaced with therapeutic genes for gene therapy applications
• Viral surface proteins can be modified to alter host cell tropism or evade immune recognition
• Reporter genes (GFP, luciferase) can be inserted into viral genomes for tracking and visualization
• Attenuated viruses can be created by deleting virulence factors or essential genes
• Genetically modified viruses can be used as vaccines, gene delivery vectors, or oncolytic agents

Viral Vectors in Biotechnology

• Viral vectors are viruses modified to deliver genetic material into target cells
• Common viral vectors include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV)
• Retroviruses and lentiviruses integrate their genetic material into the host genome, allowing stable expression
  • Lentiviruses (HIV-based vectors) can infect both dividing and non-dividing cells
• Adenoviruses and AAV do not integrate into the host genome, providing transient expression
  • AAV vectors have low immunogenicity and can target specific tissues
• Viral vectors can be used for gene delivery, protein production, or gene editing (CRISPR/Cas9)
• Packaging cell lines are used to produce high-titer viral vectors while minimizing the risk of replication-competent viruses
• Viral vectors have applications in research, biotechnology, and gene therapy

Gene Therapy Applications

• Gene therapy involves the introduction of functional genes into cells to treat or prevent diseases
• Viral vectors are the most common delivery method for gene therapy
• Monogenic diseases, such as cystic fibrosis and hemophilia, are prime targets for gene therapy
• Cancer gene therapy strategies include tumor suppressor gene replacement, suicide gene therapy, and immunotherapy
• Viral vectors can deliver genes to specific tissues or cell types, minimizing off-target effects
• Ex vivo gene therapy involves modifying cells outside the body and then reintroducing them
  • Chimeric antigen receptor (CAR) T-cell therapy is an example of ex vivo gene therapy for cancer treatment
• In vivo gene therapy involves direct delivery of viral vectors to target tissues within the body
• Gene editing technologies (CRISPR/Cas9) can be combined with viral vectors for precise genome modification

Viral Delivery Systems

• Viral delivery systems are designed to efficiently transport genetic material into target cells
• Pseudotyping involves replacing the viral envelope proteins with those from another virus to alter tropism
• Tissue-specific promoters can be used to restrict gene expression to desired cell types
• Inducible promoters allow temporal control of gene expression in response to external stimuli (tetracycline, doxycycline)
• Targeted viral vectors can be created by incorporating ligands or antibodies that bind to specific cell surface receptors
• Nanoparticle encapsulation can protect viral vectors from immune recognition and enhance delivery efficiency
• Local delivery methods, such as intratumoral injection, can minimize systemic toxicity
• Systemic delivery via intravenous injection allows widespread distribution of viral vectors

Safety and Ethical Considerations

• Safety is a primary concern in the development and application of viral vectors for gene therapy
• Replication-competent viruses pose a risk of uncontrolled viral spread and pathogenicity
• Insertional mutagenesis can occur when viral vectors integrate into the host genome, potentially activating oncogenes
• Immune responses against viral vectors or transgene products can limit efficacy and cause adverse reactions
• Rigorous testing and monitoring are required to assess the safety and long-term effects of gene therapy
• Informed consent and patient education are essential for ethical gene therapy clinical trials
• Equitable access to gene therapy treatments is an important consideration, given the high costs and limited availability
• Regulatory oversight and guidelines are in place to ensure the safety and ethical conduct of gene therapy research and applications

Current Research and Future Directions

• Ongoing research aims to improve the efficiency, specificity, and safety of viral vectors for gene therapy
• Development of non-viral delivery methods, such as lipid nanoparticles and exosomes, as alternatives to viral vectors
• Combination therapies that integrate gene therapy with other treatment modalities (chemotherapy, immunotherapy)
• Expansion of gene therapy applications beyond monogenic diseases to complex disorders (cardiovascular, neurodegenerative)
• Exploration of gene editing technologies (CRISPR/Cas9, base editing) for precise gene correction or modification
• Optimization of manufacturing processes for large-scale production of viral vectors
• Investigation of novel viral vectors, such as baculoviruses and alphaviruses, for specific applications
• Long-term follow-up studies to assess the durability and safety of gene therapy treatments

Key Experiments and Case Studies

• Successful treatment of X-linked severe combined immunodeficiency (SCID-X1) using retroviral gene therapy
• Alipogene tiparvovec (Glybera), the first gene therapy product approved in Europe for lipoprotein lipase deficiency
• Luxturna, an AAV-based gene therapy for inherited retinal dystrophy, approved by the FDA in 2017
• CAR T-cell therapy (Kymriah, Yescarta) for the treatment of B-cell malignancies
• Oncolytic virotherapy using genetically modified viruses (T-VEC) for melanoma treatment
• Hemophilia B gene therapy clinical trials using AAV vectors to deliver factor IX gene
• Leber congenital amaurosis gene therapy using AAV vectors to deliver RPE65 gene
• Duchenne muscular dystrophy gene therapy using AAV vectors to deliver micro-dystrophin gene