FINAL - CH 13 Flashcards
Ex vivo therapy:
somatic cells are removed from the patient and manipulated in the lab, then returned to the patient
In vivo therapy:
somatic cells are genetically altered in the patient
Desirable Characteristics of Somatic Cells Used for Gene Therapy
Cells are easily accessible
Long life span in body
Proliferating cells are preferred because the vector carrying the good version of the gene can integrate into new cells
Different Methods of Gene Delivery
Viral vector deliver
Liposome fusion
Electroporation
-cell is momentarily electrically shocked to create holes in the membrane that allows DNA to enter cell
CRISPR
Retroviral vectors
RNA viruses that use reverse transcriptase to make DNA copy to integrate into host chromosomes
Seldom provoke immune response
Viruses are modified to prevent replication and infection of host cells
Disadvantages for Retroviral vectors
preferentially integrates into gene-rich regions of DNA, thus can insert near a proto-oncogene and activate it to cause cancer
can only infect dividing cells because it can only integrate when nuclear membrane is dissolved.
Different Viruses used for Gene Therapy
can insert DNA into often inaccessible neurons
Lentivirus
a type of retrovirus, but can enter non-dividing cells through pores in the nuclear membrane
ex HIV
Challenges still facing viral gene therapy
Transient and low-level expression
Difficulty in reaching or specifying target tissue
Necessity for precise regulation of gene activity
Potential for insertional mutagenesis
Transient and low-level expression
only some cells incorporate the virus/gene
sometimes cells methylate, thus inactivate the gene
lack of enhancer sequences may diminish expression
Difficulty in reaching or specifying target tissue
e.g. neurons (harder) vs. lymphocytes (easier)
Necessity for precise regulation of gene activity
some conditions require strict regulation and balance of the amount of products produced
For these types of conditions (gain-of-function or dominant negative mutations), ________ therapies may be helpful:
gene blocking
Antisense therapy
Ribozyme therapy
RNA interference
gene replacement is applicable to correct _________ mutations, it is NOT effective in correcting _________ or _________ mutations
loss-of-function
gain-of-function
dominant negative
Antisense therapy
DNA oligonucleotide is synthesized to be complementary to the mRNA sequence produced by the gain-of-function mutation
This antisense DNA bind to the abnormal RNA and prevents is translation
con to Antisense therapy
the oligonucleotide is often degraded before it can reach its target
Ribozyme therapy
enzymatic RNA molecule that cuts RNA sequences
can be engineered to cut specific mRNA sequences that contain a specific mutation
RNA interference (RNAi)
natural phenomenon evolved to defend cells from viral infections
lls normally use “dicer” enzyme to digest double stranded RNA which are produced by many viruses, into 20-bp pieces. These double stranded pieces separate and are used as templates to bind to and destroy single stranded RNA with the same sequence
The RISC (RNA inducing silencing complex) helps separate the 2 strands.
can artificially synthesize double stranded RNA that correspond to disease causing DNA sequences, thereby destroying the mutant mRNA products
Although _______ is applicable to correct _________ mutations, it is NOT effective in correcting _____________ (e.g. Huntington’s, Marfan’s syndrome) because: _____________
gene replacement
loss-of-function
gain-of-function or dominant negative mutations
the defective gene product must be disabled in some way
