MIC115 Recombinant DNA Cloning Lecture 13 - Viral Vectors PDF

MIC115 Recombinant DNA Cloning 24FQ Lecture 13 – Viral vectors Options for vaccine and gene therapy Text with gray bars: you do not need to remember SUGGESTED VIDEOS Vi...

MIC115 Recombinant DNA Cloning 24FQ Lecture 13 – Viral vectors Options for vaccine and gene therapy Text with gray bars: you do not need to remember SUGGESTED VIDEOS Viral Vectors Overview https://www.youtube.com/watch?v=Q6qk6Wh6cXU The Basics of the Recombinant Lentivirus System https://www.youtube.com/watch?v=kJSsZMdA8Sk Mayo Clinic Insights: How the the Johnson & Johnson COVID-19 vaccine works https://www.youtube.com/watch?v=VnJkPL-jlFI LECTURE 13 TOPICS Viral vectors: Overview Retrovirus and Lentivirus Retroviral and lentiviral vectors Adenoviral vectors Adeno-associated viral vectors LECTURE 13 LEARNING GOALS Compare the features of viral vectors Understand the retroviral life cycle Understand the basic components of lentiviral vectors Compare the features of stable transfection vs. lentiviral transduction I. Viral vectors are used for gene delivery and gene therapy Taking advantage of viruses (viruses can deliver DNA or RNA to cells), viral vectors were developed. Viral vectors: Vehicles designed to deliver DNA or RNA directory into cells. Safety consideration: Derived from viruses, but all viral genes were removed and modified only to deliver DNA or RNA of your interests. We only use viral vectors that are extensively studied and determined to be safe for humans. Biosafety levels: we need to have a BSL-2 level facility to perform viral transduction in a lab. II. Viral vectors: Gene delivery and gene therapy 1 MIC115 Recombinant DNA Cloning 24FQ Retrovirus and Lentivirus (a type of retrovirus): ssRNA genome, they can carry larger RNAs. They will be integrated to a host genome. The difference is that retroviruses only infect mitotically active cells, while lentiviruses can also infect non-dividing and actively dividing cells. Adenoviruses have a double-stranded DNA (dsDNA) genome, and adeno-associated viruses have a single-stranded DNA (ssDNA) genome. They do not undergo genomic integration. Adenoviruses are large, but adeno-associated viruses are generally small and carry a small ssDNA genome. III. Retrovirus and Lentivirus Lentivirus is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. The best characterized lentiviral vectors are derived from HIV type 1 (HIV-1). HIV studies led to the development of lentiviral vectors IV. Retroviral and Lentiviral vectors “Transduction” means inserting the DNA into the host genome as a provirus. Retroviral vectors utilize the integration machinery of naturally occurring retroviruses to stably integrate the recombinant viral genome into a host chromosome. Retroviral vectors are useful in achieving stable and efficient transduction of a gene or genes into cells that are not easily transfected. V. Retrovirus Genome (ssRNA) The retroviral genome are located in between two Long Terminal Repeats (LTR): LTR-y (psi: packaging signal) sequence - GAG gene-POL gene-ENV gene – LTR. Essential cis elements during retroviral particle production: The Long Terminal Repeats (LTRs) is required for: integration of proviral DNA, initiation of viral genomic RNA synthesis. The y (psi: packaging signal) sequence is required for viral packaging. VI. Retrovirus Virion (A complete virus particle) RNA genome: Each virion contains two identical single-stranded RNA molecules. Major genes in the retroviral genome encodes major structural components. 1. GAG gene: The core of the virion is encapsidated by the virus structural proteins encoded in the GAG gene, forming the nucleocapsid. The nucleocapsid is surrounded by a lipid bilayer derived from the plasma membrane of the host cell. 2. POL gene: The POL gene encodes a reverse transcriptase. 3. ENV gene: The ENV gene encodes the glycoproteins inserted in this membrane. 2 MIC115 Recombinant DNA Cloning 24FQ These retroviruses already occupy our human genome (5% of the human genome consists of “endogenous” retrovirus)! VII. Retroviral Life Cycle (key steps) Membrane fusion Reverse transcription (the RNA genome becomes the dsDNA genome) Integration to a host genome Transcription and assembly using the host machinery Packaging using the plasma membrane of the host cell Key facts of the retroviral life cycle: Viral tropism: the ability of a given virus to productively infect a particular cell. Determined between “envelope glycoprotein” and “cellular receptors on the host cell surface” (e.g. HIV to specific CD4 receptors on helper T-cell lymphocytes). Integration: The integration of the double-stranded proviral DNA is a random process. LTRs are necessary for integration. Many retroviruses require mitosis for productive infection because the pre-integration complex cannot pass through nuclear pores. Lentiviruses, a subtype of retrovirus, can infect both dividing and non-dividing cells because certain viral proteins facilitate the active import of the pre-integration complex into the nucleus. VIII. Viral Vectors Introduce recombinant DNA into vertebrate cells by retroviral/lentiviral vectors Most viruses are pathogenic because they can propagate! However, replication-incompetent viral vectors can be derived from certain viruses to transfer genes to animal cells. (this is achieved with separate plasmids). To produce infectious viral particles containing the transgene, the viral structural proteins (encoded by gag, pol, env, and rev) All the genes encoding virulent viral factors are deleted to make them safe to use. Recombinant Lentiviral Packaging System Key points: Removed toxic viral genes and split essential viral genes to separate plasmids Quiz: Which plasmid is packaged to viral particles? A: transfer plasmids because it has the y sequence Quiz: If you move the y sequence from the transfer plasmid (which contains the transgene) to other plasmids, can you package the transgenes? A: No, because the y sequence is necessary for packaging. 3 MIC115 Recombinant DNA Cloning 24FQ Key points: Recombinant Lentiviral Packaging System 1. To produce infectious and replication-incompetent viral particles containing the transgene, the viral structural proteins are supplied with the separate plasmids. 2. 2. The y (psi: packaging signal) sequence is required for viral packaging. A transgene plasmid must have the y sequence. Discussion question: You want to express a luciferase gene in human cells using a retroviral vector. After making replication-incompetent retroviruses carrying the luciferase gene, you infect the target cells with the retroviruses and monitor luciferase gene expression every day. You see a different expression pattern (solid line) compared to the transient transfection experiment (dotted line). Why? A: This is because the retroviral infection causes genomic integration, but transient transfection does not. Discussion: Compare the features of stable transfection vs. lentiviral transduction IX. Pseudotyping retroviral vectors with VSV-G Replace Env glycoprotein with Vesicular Stomatitis Virus Glycoprotein (VSV-G) to widen the range of cell types the viral vectors can bind to. VSV-G interacts with a phospholipid component of the plasma membrane to mediate infection. Virus entry is not dependent on the presence of specific protein receptors X. Limitations of the retroviral vectors Retroviral transduction may not be the method of choice when working with cells that can be efficiently transfected or in experiments in which high transgene expression level is desirable. Because the size of retroviral RNA that can be efficiently packaged is limited, retroviral gene transfer is unable to transduce large (>10 kb) DNA fragments. XI, Adenovirus Does not integrate into host genome 4 MIC115 Recombinant DNA Cloning 24FQ Adenoviruses have dsDNA genomes. Adenoviruses are large. Remains in the nucleus as episome. Adenovirus vectors: Adenovirus vectors are widely used for vaccines and in vivo gene therapies because they do not induce genome integration (safe and efficient!) Used for the COVID vaccine Do not need to remember other details. XII. Adeno-associated virus (AAV) Adeno-associated virus (AAV) has a small ssDNA genome: 4.7kb genome Does not integrate into host genome AAV vectors: Stably maintained in non-dividing target cells (liver, nerve system, etc). Popular tools for in vivo gene therapies. Do not need to remember other details. 5