BME 6210 E3 TEST WITH
COMPLETE SOLUTION
Main Nucleic Acid Delivery Types - ANSWER DNA
siRNA
mRNA
miRNA
(m)DNA - ANSWER Mutated DNA
Leads to Mutated RNA (m)mRNA
Leads to Mutated Protein (m)Protein
Nucleic Acids as Therapeutics - ANSWER Want to transform (m)Prot into Prot
Characteristics of dNTPs - ANSWER -Big (1000+ MW): to big for passive
transport
-Negatively charged with a phosphate backbone: too hydrophilic to passively
pass phospholipid bilayer
-Susceptible to enzymes: As dNTPs shouldn't be outside of cell, nuclease eats
them.
Characteristics of Ideal DNA Delivery System - ANSWER 1: Condense and
encapsulate DNA
2: Bind package to cell (R-L or non-specific)
,3: Needs to be internalized by cell (endocytosis)
4: Escape from internalized vesicle/endosome so it doesn't get degraded in
the lysosome
5: DNA is transported to nucleus or RNA in cytosol
5a: DNA complex is disrupted and DNA is delivered to nucleus
Viral Vectors - ANSWER Used because they are designed to deliver dNTPs
Just need a bit o' tweaking
Types of Viral Vectors - ANSWER Adenovirus - delivers DNA, used clinically
Retrovirus - delivers RNA, used clinically
Adeno-Associated Virus (AAV) - delivers DNA, non-clinical
Enti-Virus - might deliver DNA, used in labs
Replication Deficient - ANSWER Virus cannot replicate by itself and take-over
Limits of Viral Vectors - ANSWER Can only hold so much DNA, so early viral
genes are removed
Removed Viral Genes - ANSWER E1: Required for replication
E3: Not required for infection (unnecessary for therapy)
Manufacturing virus - ANSWER HEK 293 is infected with E1 so HEK293 can
replicate virus
As virus doesn't have E1, it can't replicate itself
(m)HEK 293 grown in bioreactor to mass produce virus
Virus can be purified from HEK 293 to make vaccine
, Viral Vector Advantages - ANSWER -High efficiency with 40% vectors making
it to nucleus
-Can infect replicating and non-replicating cells
Viral Vector Disadvantages - ANSWER -Limited therapeutic gene size as
vector has upper limit
-Cause immune response (limited dosing before memory T-Cells)
-Potential to activate and replicate oncogenes (retroviruses may encode
oncogenes)
Non-Viral Vectors - ANSWER Synthetic vectors utilized to transfer DNA into
cells for gene therapy purposes
-Ca3(PO4)2
-Lipids
-Polycations
Calcium Phosphate - ANSWER Ca3(PO4)2
Water insoluble (hydrophobic) at pH 7 so encapsulate DNA (hydrophilic)
Formed with CaCl + NaCl
Hydrophobic so can be internalized
Inefficient
Lipids - ANSWER - Idea: Liposomes (+charge) will encapsulated DNA (-
COMPLETE SOLUTION
Main Nucleic Acid Delivery Types - ANSWER DNA
siRNA
mRNA
miRNA
(m)DNA - ANSWER Mutated DNA
Leads to Mutated RNA (m)mRNA
Leads to Mutated Protein (m)Protein
Nucleic Acids as Therapeutics - ANSWER Want to transform (m)Prot into Prot
Characteristics of dNTPs - ANSWER -Big (1000+ MW): to big for passive
transport
-Negatively charged with a phosphate backbone: too hydrophilic to passively
pass phospholipid bilayer
-Susceptible to enzymes: As dNTPs shouldn't be outside of cell, nuclease eats
them.
Characteristics of Ideal DNA Delivery System - ANSWER 1: Condense and
encapsulate DNA
2: Bind package to cell (R-L or non-specific)
,3: Needs to be internalized by cell (endocytosis)
4: Escape from internalized vesicle/endosome so it doesn't get degraded in
the lysosome
5: DNA is transported to nucleus or RNA in cytosol
5a: DNA complex is disrupted and DNA is delivered to nucleus
Viral Vectors - ANSWER Used because they are designed to deliver dNTPs
Just need a bit o' tweaking
Types of Viral Vectors - ANSWER Adenovirus - delivers DNA, used clinically
Retrovirus - delivers RNA, used clinically
Adeno-Associated Virus (AAV) - delivers DNA, non-clinical
Enti-Virus - might deliver DNA, used in labs
Replication Deficient - ANSWER Virus cannot replicate by itself and take-over
Limits of Viral Vectors - ANSWER Can only hold so much DNA, so early viral
genes are removed
Removed Viral Genes - ANSWER E1: Required for replication
E3: Not required for infection (unnecessary for therapy)
Manufacturing virus - ANSWER HEK 293 is infected with E1 so HEK293 can
replicate virus
As virus doesn't have E1, it can't replicate itself
(m)HEK 293 grown in bioreactor to mass produce virus
Virus can be purified from HEK 293 to make vaccine
, Viral Vector Advantages - ANSWER -High efficiency with 40% vectors making
it to nucleus
-Can infect replicating and non-replicating cells
Viral Vector Disadvantages - ANSWER -Limited therapeutic gene size as
vector has upper limit
-Cause immune response (limited dosing before memory T-Cells)
-Potential to activate and replicate oncogenes (retroviruses may encode
oncogenes)
Non-Viral Vectors - ANSWER Synthetic vectors utilized to transfer DNA into
cells for gene therapy purposes
-Ca3(PO4)2
-Lipids
-Polycations
Calcium Phosphate - ANSWER Ca3(PO4)2
Water insoluble (hydrophobic) at pH 7 so encapsulate DNA (hydrophilic)
Formed with CaCl + NaCl
Hydrophobic so can be internalized
Inefficient
Lipids - ANSWER - Idea: Liposomes (+charge) will encapsulated DNA (-
