Unit 2a: AAV Vectors & In Vivo Gene Therapy Flashcards
What is gene therapy?
The introduction of nucleic acids into cells for the sake of altering the course of a medical condition/Treating diseases by transferring a normal, functioning copy of the gene into patient’s cells
What is ex vivo/In vivo
Ex vivo: modifying the gene outside the body and then inserting it
In vivo: directly inserting DNA into patient’s cells (i.e. viral vectors)
What machinery do viruses have and have not
Viruses have genetic information (DNA or RNA) but do not have the machinery to carry out tasks of making RNA or a Protein, instead, they depend on the host cell to do it.
How can viruses be used to target specific types of cells or areas of the body
Many viruses can only affect specific types of cells and it has to do with the protein shell of a virus that binds to proteins that can only be found on the surface of certain target cells.
Where does AAV DNA stay inside the cell?
AAVs can be contained in the nucleus outside of the chromosome which means they don’t touch and don’t integrate into the DNA genome of the host cell which makes their DNA Extrachromosomal.
What is the potential danger of inserting the viral DNA into the chromosome?
if you insert a piece of DNA into a chromosome you might insert it into an important gene and affect its function/insert DNA into the DNA and turn on a gene that shouldn’t be turned on, etc
What is a Capsid and what is its function?
Capsid - the protein shell of a virus, enclosing its genetic material, a virus’s ability to infect the cell depends on the capsids that it has (capsid’s surface proteins that are going to encounter the cell).
A primary function of the capsid is to protect the viral genome from environmental conditions and ultimately to deliver the genome to the interior of a homologous host cell.
What are serotypes?
Serotypes are AAV capsid proteins
What is CAP, REP, ITRs and what functions do they serve?
CAP is a gene for viral capsid protein
REP is a gene for DNA replication
IRTs are inverted terminal repeats that are required for efficient DNA replication
ITRs have to be in the vector, but rep and cap don’t
What are the stages of viral infection?
- The virus just floats around and when it sees a protein on the surface of a cell it binds to it
- Then the cell recognizes it (called endocytosis, a cellular process in which substances are brought into the cell.) and brings in a piece of the plasma membrane.
- The plasma membrane then undergoes invagination (the process of being turned inside out or folded back on itself to form a cavity or pouch).
- Then the membrane pinched off the plasma membrane (called endosome) surrounds the virus and eventually breaks down
- Virus moves into the nucleus from the broken membrane
- Once it’s inside the nucleus the virus undergoes DNA replication using the ITRs
The whole process is blind to the DNA and done purely by the capsid of the virus
How do we make an AAV useful in gene therapy?
To make a virus useful we replace the viral DNA (rep and cap) with therapeutic DNA together with a gene regulatory sequence that tells the gene what cells to bind to. A virus can target many cells but we want to make it bind to specific desired cells only. Once we put the therapeutic DNA instead of rep and cap the virus cannot reproduce and make daughter viruses
What are the disadvantages of using an AAV vector?
- In dividing cells the viral DNA can be lost through cell replication as extrachromosomal is not replicated along with the host cell DNA, yet some viral vectors will cause the gene they are carrying to be integrated into a cell’s genome which helps ensure that the gene is not lost when the cells divide.
- The cloning capacity is limited to 4800 base pairs which makes big genes invalid for the AAV gene therapy
- Even if an AAV vector successfully delivers a genome to the cells it doesn’t guarantee the production of the desired protein because it requires a promoter and proper transcription factors in the cells to recognize that promoter and drive the expression of the gene it controls
What are the 3 DNAs put into an rAAV vector?
- Transgene (therapeutic gene of interest) which replaces both cap and rep
- Rep and cap genes are provided in trans on a “helper” plasmid that cannot be incorporated into an AAV particle
- Adenoviral components which are provided by the transfection of a third plasmid and have other helping functions
What do you need to make protein from an AAV vector?
A promoter which is a gene regulatory sequence
What is Recombinant DNA?
Recombinant DNA molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome
