MCB4503 Module 11
true or false: scientists develop a virus that replicates in the cancer cells that have high concentrations of a certain enzyme. this means the virus can only infect and replicate in this type of cancer cell
false; can infect both normal and cancer cells, but can only replicate in cancer cells
true or false: all oncolytic viruses target the tumor cell
false; can target the tumor cell or its microenvironment
maturation of adenoviruses occurs where? maturation of HIV occurs where?
inside nucleus; HIV occurs after the virus has budded off from the host cell
what is gene therapy
involves correcting a genetic disorder by delivering a functional gene to replace the function of a missing or nonfunctional gene in a cell
most of the viruses that are used to produce toxic enzymes that will kill the host cell (cancer cells) are typically
large dsDNA viruses like poxviruses or herpesviruses (has enough space for genes that produce toxins)
the first oncovirus approved for cancer therapy was ? what kind of oncovirus was it?
modified virus called T-VEC (directly injected to treat melanoma)
which virus randomly integrates into the host's chromosome
retrovirus
___ will soon overtake adenoviruses as the most commonly used vectors in gene therapy treatments
retroviruses (like HIV)
a major advantage of the use of oncolytic viruses is that
their toxicity is limited
a disadvantage of oncolytic viruses is that
they have limited potential as a monotherapy of its own (not very effective alone, needs to be combined w other treatments)
what is the primary vector used to deliver functional genes to target cells
viruses
what are oncolytic viruses
viruses that lyse cancer cells
human stem cells have homing properties. what does this mean
you can inject them into a person and they will go to the tissues where they're supposed to be
what is AAV, what is its structure
- adeno-associated virus is a helper-dependent parvovirus - one of the smallest non-enveloped capsid viruses - small, linear, ssDNA genome w inverted terminal repeats - genome consists of only 2 genes: viral replication gene and capsid gene - these genes encode all non-structural and structural proteins - does not encode own polymerase, uses cell's
what approach do most oncolytic viruses take
- cause lysis (disintegration and rupture) of tumor cells - stimulate innate or adaptive immunity through viral and tumor antigens
describe the infectious cycle of an adenovirus
- cycle begins when protruding fibers bind to coxsackie-adenovirus receptors (CARs) on host - virus moves along surface until it finds a clathrin-coated pit; enters through clathrin-mediated endocytosis - now inside the endosome, the protrusion are gone - fiber base (penton) interacts w cell proteins (integrin) to disassemble the capsid - virus uncoats, genome transported to nucleus - host cell polymerase transcribes E1A; mRNAs splices and transported to cytoplasm, make proteins that then go back to the nucleus for expression regulation - at same time, host cell polymerases also transcribe virus associated genes; resulting proteins go to nucleus - both viral and cell proteins used to copy viral genome; genome can then transcribe late genes or be used as a template for replication - late genes encode structural proteins, which go into new viral particle - virus matures after the capsid is enclosed, and the viral proteins inside cleave other viral proteins
what are the methods of viral vector delivery in gene therapy? briefly describe them
- direct delivery: direct injection of viruses into tissue - cell-based delivery: involves removal, infection, and reinsertion of patient's primary somatic cells
what is the RAS oncogene
- found in many cancers - when activated, it triggers the production of more cathepsin, a host protein that assists in viral uncoating - at the same time, it blocks PKR, a protein kinase that inhibits translation of the viral protein - RAS oncogene good for viral uncoating and translation
what is the main difference between gene therapy and oncolytic viruses?
- gene therapy goal is to keep the human cells alive by delivering a functional gene - oncolytic virus goal is to infect and kill cancer cells to destroy the tumor
what are the characteristics of viral vectors used in gene therapy
- genome capacity: amount of genetic space available to incorporate gene of interest - cellular tropism: types of cells the virus infects - stability of expression: length of time the gene is expressed in the cell after the virus delivers it - infection efficiency: how quickly target cells are infected, weakens immune response - pathogenicity: capacity to cause disease should be low (trying to cure disease!) - immunogenicity: ability to avoid immune response and deliver gene of interest - variety of serotypes: many virus variations stops a person from developing immune response to 2nd serotype - resting cell infection: may need a virus that can replicate in a cell that isn't actively dividing (like a brain cell) - ease of propagation: virus must be easy to grow and purify
how is the adenovirus genome manipulated to make it a better vector?
- have prevented the virus from replicating by removing the E1A gene (thus, virus can infect and deliver genome, but not replicate) - have removed unnecessary genes to make room for therapeutic genes (changing length could disrupt the infectious cycle, so genes like E3 not essential to early infection are removed to make room for therapeutic gene)
what are some of the disadvantages of retroviruses in gene therapy
- integration can lead to transformation and tumor formation - difficult to propagate (difficult to make in highly concentrated stocks) - low capsid stability (might degrade) - usually only infect dividing cells - mild pathogenicity
advantages of adenoviruses as viral vectors for therapy?
- large genome capacity (have large, dsDNA genomes that can hold genetic material) - can infect non-dividing cells - rapid gene expression - do not integrate into host chromosome (reduces possibility of a genetic mutation, which could lead to transformation and tumor formation) - high ease of propagation (can be easily purified and concentrated) - many serotypes (no interference from immune response) - broad cell tropism (can target many cells)
disadvantages of adenoviruses as viral vectors for therapy?
- mild pathogenicity (can infect tonsils, especially in those w weak immune systems) - rapid immune response (virus quickly cleared) *do not integrate into host chromosome (usually an advantage, can be a disadvantage too bc not all daughter cells will have the virus/gene of interest, so multiple doses of treatment are needed)
what are some of the advantages of retroviruses in gene therapy
- moderately large genome (not as large as adenoviruses) - high infection efficiency (less likely immune system will clear them) - stable, long term gene expression - many pseudotypes available (a serotype that doesn't occur naturally) - low inflammatory response
what is T-VEC, specifically its role in gene therapy
- modified virus, first oncovirus approved for cancer therapy - modified herpes simplex 1 virus - has had 2 viral genes removed and 1 gene added back in - ICP34.5 (needed for replication in normal cells only) and ICP47 (suppresses immune response) were removed - GM-CSF (cytokine that promotes recruitment and maturation of dendritic cells, leading to tumor-specific T cells) added in
how can viruses by engineered to target diseases
- modifying viruses to specifically infect certain cell types - modifying viruses to selectively replicate in certain cells (ie. replicate in cells w high titers of a certain enzyme) - using viruses as delivery vectors (carry genes that modify or replace defective ones in host, or kill host cell) - altering the extracellular environment, which can kill target cells
what are the advantages of AAV in gene therapy?
- no pathogenicity (bc it can't replicate on its own, needs a co-infector for active infection) and low immunogenicity - many available serotypes w different cellular tropisms - infects both dividing and non-dividing cells - long term gene expression by integrating specifically into the host chromosome (cell transformation and tumor formation risk low)
describe the adenovirus genome, including which genes are expressed
- non-enveloped, dsDNA - fibers protrude from 5 fold axes of symmetry, role in binding to receptor - first viral gene transcribed is E1A gene, which causes cell to remain in DNA synthesis phase - gene specifically produces a protein that promotes transcription of viral genes, and regulates growth cycle of host cell - E3 gene is expressed later, no role in early infection (removed in therapy)
what is the VEGF protein
- produced by tumor cells to stimulate the growth of nearby blood vessels - can be blocked through genetic modifications; this targets the tumor microenvironment instead of the tumor cell itself
how does direct delivery work in gene therapy
- put a therapeutic gene into viral genome - produce viruses in culture and purify stocks - directly deliver virus into infected individual
viruses can be engineered to target diseases by altering the extracellular environment. these changes can kill target cells. how?
- reducing angiogenesis: inhibits formation of new blood vessels, starving the cell of nutrients needed to grow; doesn't usually kill cell but reduces its size and slows progression - stimulating an immune response: virus triggers cells to secrete molecules that activate the immune system and target cancer cells
what is a serotype? which serotype do we want for gene therapy?
- refers to a distinct variation within a virus that elicits the production of a distinct set of antibodies - want multiple serotypes - thus, if a person develops an immune response to one serotype, he/she won't be able to defend against another serotype
an example of a wild type oncolytic virus is a ___. what is the structure of the genome? how do these specific viruses work?
- reovirus, dsRNA virus that infects gastrointestinal tract - targets tumors w an activated RAS oncogene (this provides good environment for viral uncoating and translation) - reovirus induces apoptosis to kill the cell - usually combined w other treatments - pro: limited pathogenic effect on host - con: almost everyone has been infected w this already, so immune system clears it easily
describe the history of oncolytic viruses
- since 1800s, scientists have observed tumor regression (smaller) after recovery from viral infections (flu or chickenpox) - 1900s, unmodified viruses used to treat cancer - these trials were high risk and deemed a failure bc the viruses were not modified at all, and alternative cancer treatments (radiation, chemotherapy) started to be used w better results - radiation, surgery, and chemotherapy deemed 3 pillars of cancer treatment
what are the disadvantages of AAV in gene therapy?
- small genome capacity (restricts size of genes it carries) - low ease of propagation (difficult to produce in culture, doesn't grow in high titers/concentrations, hard to produce enough at a sufficient purity)
how do oncolytic viruses selectively hijack cancer cells, rather than healthy cells?
- tumor cells have many of the right receptors for viral entry - tumor cells divide rapidly, which supports increased replication of viruses (tumor cells select for viral activity) - many tumor cells lack antiviral defenses and immunity (easy for viruses to infect these and replicate)
how does cell based delivery work in gene therapy
- used to target cells not easily infected through direct injection (stem cells or cells in bone marrow) - therapeutic gene inserted into viral genome, cultured, and purified - primary cells are removed and cultured - these cells are infected w virus; therapeutic gene delivered - once majority of culture have this gene, cells are injected back into patient
oncolytic viruses can be divided into two types. what are they, and briefly describe each
- wild type: viruses that have not been modified in any way - modified: most oncolytic viruses, usually modified to infect both normal and cancer cells, only replicates in cancer cells
first viruses ever used in gene therapy treatments? most common viral vectors used?
adenoviruses (for both)
