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Biopharmaceutics > Term 1 > Flashcards

Term 1 Flashcards

1
Q

applications of biotechnology

A

AB production, Ab production, transgenic animals, gene therapy, vaccines, protein pharmaceuticals

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2
Q

do animal growth hormones have therapeutic value in humans

A

no

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3
Q

what happened when cadaver isolated growth hormones were used? What was done?

A

transfer of diseases such as creutzfeldt jacob disease and a shortage in availability. In 1980s, human growth hormone was produced using bacterial cells

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4
Q

pharmacists’ role in biotech

A

1) product evaluation and selection 2) patient education and counselling 3)provision of drug info 4) assistance in patient monitoring 5) drug control and prep

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5
Q

what plant cells have that animals don’t

A

cell wall, chloroplasts, and centriole

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6
Q

what has DNA/nucleus

A

chloroplasts, mitochondria and and nucleus all have DNA and nucleus (all double membraned)

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7
Q

how many nucleotides are in the human and mouse genome?

A

3 x 10^9

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8
Q

A pairs with

A

T (2 bonds)

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9
Q

G pairs with

A

C (3 bonds)

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10
Q

do introns or exons code for protein

A

exons

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11
Q

do higher organisms have more junk DNA (non coding, or “intron” regions)?

A

yes (only about 1.5% codes for proteins)

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12
Q

mRNA

A

duplication of genetic material

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13
Q

tRNA

A

translation of genetic material

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14
Q

rRNA

A

site for protein synthesis on ribosomes

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15
Q

what differs in RNA in base paits

A

U instead of T

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16
Q

nucleotide contains

A

sugar, phosphate, base

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17
Q

where does protein synthesis occur

A

cytoplasm

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18
Q

what enzyme initiates transcription

A

RNA polymerase

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19
Q

start codon

A

AUG

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20
Q

order of translation

A

initiation, elongation, termination

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21
Q

stop codons

A

UAA, UAG, UGA (has to be U and then at least one A)

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22
Q

there is at least one unique tRNA for each of the ___ (#) AAs

A

20

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23
Q

how to sequence DNA (what must we start with)

A

primer**, need before enzymes work

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24
Q

what kind of DNA do bacteria have

A

both plasmid and genomic

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25
Q

why manipulate plasmids?

A

easy to grow and maintain bacterial cells, plasmid DNA is easy to isolate

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26
Q

3 important sections of plasmid

A

promoter, reporter gene, multiple cloning site

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27
Q

2 key enzymes that made it possible to manipulate sequences of plasmids

A

restriction endonucleases (cut DNA by recognizing specific BPs), and DNA ligases (link DNA fragments)

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28
Q

what was the first biopharmaceutical

A

insulin

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29
Q

phases of bacterial growth

A

lag, log, stationary, death

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30
Q

explain lag phase

A

number of bacteria does not change with time

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31
Q

explain log phase

A

number of bacteria increases exponentially

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32
Q

explain stationary phase

A

no net change in number of bacteria- grow and divide at same rate and die. Happens when overcrowded and nutrients are depleting)Most important biological products (especially secondary metabolites like ABs) are produced here

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33
Q

explain death phase

A

bacteria decrease with time

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34
Q

any protein can be produced using genetically engineered organisms, but not every type of protein can be produced by any cell type

A

true

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35
Q

bacteria are not capable of post translational modifications

A

true- cannot glycosylate, etc

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36
Q

limitations to using bacterial expression systems

A

no post translational modification (mammalian can), proteolytic cleavage can degrade product, must break/lyse cell to get protein out (are secreted into media in mammalian cells)

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37
Q

animal cells have limits to dividing. How do you overcome this

A

hybridomas (genetically engineered animal cells with no limits on dividing- this is what cancer cells produce)

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38
Q

other name for bioreactor

A

fermentor (type where biocatalyst is a living cell)

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39
Q

step one in production of biopharms (synthesis)

A

cell culture started in small bottles, then small bioreactor, eventually large

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40
Q

types of cultivation systems

A

-free in suspension, -attached to microspheres or entrapped in matrices that are usually solidified agar, -or immobilized state as monolayers)

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41
Q

types of bioreactors

A

liquid (aka submerged) (most-saves space and more ammmenable to design, cost effective, easier to maintain, don’t have to worry about monolayers or microspheres are free in suspension)) or solid state (surface)

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42
Q

monitoring of bioreactors

A

temperature, sufficient substrate (usually carbon source), sugars/proteins.lipids, water, salts, vitamins, oxygen, optimum pH, product and by product removal

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43
Q

types of submerged (liquid) bioreactor operation types

A

batch, continuous, fed-batch

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44
Q

forms of biocatalyst for submerged (liquid) bioreactors

A

free cell (enzyme), immobilized cell (enzyme) (packed bed, membrane reactor)

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45
Q

bioreactor systems

A

stirred tank, airlift, microcarrier (ie fixed bed reactors)

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46
Q

stirred tank bioreactors

A

-air enters at bottoms, baffles range from 4-8, key factors controlled are pH/temp/oxygen, mixing method is mechanical agitation, requires energy input, bubbles are reduces by an agitator, exhaust gas flows out top

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47
Q

baffle purpose

A

used to reduce vortexing

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48
Q

what is mixing necessary for

A

maintain homogeneity, attain rapid dispersion and mixing of components injected into the fermentor, enhance heat transfer and temp control, enhance mass transfer

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49
Q

airlift bioreactors

A

reaction medium kept mixed and gassed by air or another gas at the base of reactor, seperated into gassed and ungassed regions generating a vertically circulating glow

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50
Q

advantages of airlift bioreactor

A

simple, no moving parts, less maintenance, less risk of defects and easier sterilization, lower shear rate, both plant and bacterial cells, well controlled flow and efficient mixing, large volume tanks possible which increases the output, enhances oxygen solubility achieved in large tanks with greater pressures, greater heat removal

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51
Q

disadvantages of airlift bioreactor

A

higher initial investment, greater air and higher pressures needed, impossible to maintain consistent levels of substrate, nutrients and oxygen with circulation and changing conditions, inefficient gas and liquid separation when foaming occurs

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52
Q

micro carrier bioreactors

A

can have extremely high productivity within a compact size, used for culture of immobilized mammalian cells, us porous glass beads to give large SA,

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53
Q

batch operations

A

everything put in vessel together, let it grow, watch for products, harvest. Once started, no inlet or outlet unless aerobic system and gas inlet and outlet should be there. Between batches, down time to harvest, empty, clean, sterilize and refill- lowers efficiency)

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54
Q

continuous operation

A

don’t add substrate all at once, remove throughout. Continuous medium flow- incoming stream (feed) contains substrate and leaving stream (effluent) contains product. Good to maintain bacteria in log phase (keep there longer), contamination more likely

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55
Q

2 assumptions with continuous operations

A

mixing is vigorous (concentrations are uniform throughout the reactor), effluent concentration is same with reactor contents (chemostat=continuous configurations for cultivation of microorganisms) (conditions don’t change through time=steady state-no accumulation of components ie too much feed, and no depletion of source ie too much effluent)

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56
Q

advantages of batch

A

-most commonly used because reduced contamination or mutation (due to relatively brief growing period), lower investment, more flexible, higher raw material conversion, easy to operate

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57
Q

disadvantages of batch

A

lower productivity (maintenance) (down time), increased focus on instrumentation, greater expense in preparing several subcultures for inoculation, higher labor and process control costs, batch to batch variability, accumulation of inhibitory products

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58
Q

continuous reactors advantages

A

increased opportunities for system investigation and analysis, higher degree of control, results are more reliable and reproducible (quality), efficient, higher productivity, uniform product, no accumulation of inhibitory proteins

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59
Q

disadvantage of continuous reactors

A

higher contamination and cell mutation risk (long growing periods and continuous reactions, higher investment, less flexible

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60
Q

fed batch

A

modification of batch cultivation in which nutrient is added intermittently

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61
Q

cell bank (and 2 types)

A

when cell line is to be used over many cycles- master cell (expanded to WCB) and working bank (ones currently use and experiment with)

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62
Q

cell banks must be tested for

A

contaminents, and some for viruses

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63
Q

higher speed centrifuge is required for separation of smaller particles

A

true

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64
Q

inclusion bodies

A

bacterial proteins aggregate - often occur when bacterial cells overproduce proteins through the use of plasmid expression

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65
Q

advantages and disadvantages of inclusion bodies

A

A- relatively simple to recover, protected from proteolytic cleavage D- biologically inactive- must be denatured first to be solubilized then refolded for activity, inaccurate assessment of recovery-can’t directly measure, recovery of proteins requires cell breakage/protein sedimentation/pellet washing.

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66
Q

disrupting cells can be 2 ways

A

-mechanical (sonication and liquid shear homogenization) and non (autolysis, osmotic shock)

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67
Q

if proteolytic degradation will occur, purification should be carried out at ___degrees celsius

A

4

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68
Q

inclusion bodies dissolve in denaturing agents such as

A

SDS, urea, guanidine HCl

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69
Q

if molecules are interlinked by disulfide bonds, must add ___ agents such as

A

reducing- mercaptoethanol, dithiothreitol (DTT)- they may enhance or facilitate the solubilization of protein inclusion bodies

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70
Q

presence of glycosylation can affect

A

solubility, stability, serum half life, pharmacological fx, immunogenicity

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71
Q

protein glycosylation is directly determined by DNA sequence

A

false

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72
Q

why can’t most proteins be sterilized by the standard methods

A

denaturation at high temperatures

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73
Q

components of parenteral formulations

A

solubility enhancers, anti adsorption and anti aggregation agents, buffer, antioxidants, preservatives, osmotic agents, carrier systems, active ingredient

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74
Q

why are solubility enhancers needed in parenteral formulations

A

generally a high concentration is used in biotech drugs, and this can cause proteins to aggregate and precipitate out (lowers potency, increasing change of immunogenicity). Interaction with excipients instead of other proteins= MOA

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75
Q

when does aggregation of proteins occur

A

hydrophobic or electrostatic interactions between different proteins

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76
Q

approaches to enhance solubility

A

proper pH and ionic conditions, additions of AA like lysine and arginine (tend to solubilize t-PA), adding surfactants (SDS), sugars like glucose and sucrose

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77
Q

what is used to prevent insulin adsorption

A

albumin- has higher affinity for surfaces (competition)

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78
Q

what is likely to form fibrillar precipitates

A

(long rod shaped structures); low concentrations of phopholipids and sufactants, insulin; solve by selecting proper pH often

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79
Q

common buffer system components in biotech

A

phosphate, citrate, acetate

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80
Q

when freezing conditions (lyophilizing)- buffers can ___ or form _____ and pH changes will be minimized

A

crystalize (Na2 faster than Na), or don’t crystalize- form amorphous systmes

81
Q

what is most often oxidized

A

AA

82
Q

how to add an antioxidant

A

replacement of O2 by inert gases helps reduce oxidative stress, special airtight vials, vitamins A,C,E, AAs, chemicals (selenium, acetic acid, sodium citrate)

83
Q

preservatives- what to take into consideration

A

lowest concentrations possible to minimize SE, want as not toxic and immunogenic as possible

84
Q

preservatives-bacterio static or cidal

A

ususally static- stop growing not kill

85
Q

what do osmotic agents do

A

modulate tonicity; can use preferential exclusion to stabilize proteins (osmotic agent increases interaction of protein with solvent so that it is in the solubilized state not precipitating out) (they themselves are excluded from protein surface layer) (puts protein in solubilized state longer)

86
Q

problem with strong preferental exclusion

A

enhances tendency of proteins to self associate (add osmotic agents to enhance interaction of solvent with protein and they themselves are excluded from protein surface layer)

87
Q

what is product purity

A

greater than or equal to 99%

88
Q

using serum may do what

A

introduce contaminating proteins (especially fetal calf serum-often used in mammalian cell cultures), can have viruses/bacteria/prions/fungi, even when sterile may contain endotoxin (should be eliminated during purification)

89
Q

contaminants present in recombinant protein products from bacterial and non sources are 1 of 3 things

A

host related, product related, or process related

90
Q

most frequent source of virus introduction- how to solve

A

animal serum- can also introduce bacteria, mycoplasmas, fungi and endotoxins- move towards defined growth media where serum levels are significantly reduced

91
Q

how are viruses inactivated (physical and chemical) or removed

A

heat, irradiation, sonication, extreme pH, detergents, solvents, certain disinfectants. Removed= chromatography, filtration, precipitation

92
Q

how to remove bacteria from product

A

size allows simple sterile filtration

93
Q

how to remove pyrogens

A

not by sterile filtration, usually at least one step of ion exchange chromatography (anionic exchange) to remove the negatively charged pyrogens

94
Q

what are pyrogens and what do they cause

A

usually endotoxins of gram negative bacteria, causes strong fever and can be fatal

95
Q

methods to detect if cellular DNA is present in our final product (we want it removed)

A

radiolabelled nucleotides, dye binding

96
Q

why are protein contaminants a problem

A

may be recognized as antigens by patient and on repeated use may show an immune reaction- can be misinterpreted as the protein of interest we want to cause benefit causing this immunogenicity

97
Q

what are sources of protein contaminants

A

growth medium, host proteins of cells (which are similar in structure and might be co purified with desired product), ligands from affinity columns used in the purification process

98
Q

serum free media is favored for large scale manufacturing for pharmaceutical proteins

A

true

99
Q

barriers to extensive use of serum free media

A

insufficient cell growth, productivity, cells grow slower

100
Q

what is often the rate limiting step in development of a biotech pharmaceutical

A

chromatography- usually 2-3 different chrom procedures will be used in succession to isolate the protein of interest

101
Q

phases of chromatography

A

stationary (insoluble matrix), mobile phase (solution passed through or over stat phase), separation based on differences between two phases- goal is to elute proteins off column one at a time

102
Q

direct immunofluorescence

A

add fluorescent body to antigen- greater binding will give greater fluorescence. Can check for type and quantity of protein. Very expensive

103
Q

indirect immunofluorescence

A

attach antigen but use unlabled antibody not fluorescence tagged. Then use common antibody against all immunoglobulins (ie all antibodies) with fluorescence tag

104
Q

difference between immunofluorescence and ELISA

A

immuno detects fluorescence, ELISA detects substrate

105
Q

inderect ELISA

A

give antibody, binds antigen, add antibody linked to enzyme, substrate converted to product- more enzyme=more color=more antigen

106
Q

steps in ensuring biotech quality

A

plasmids and host cells, protein stability, process validation (for purification), final product batch check

107
Q

stability testing of final labeled products include

A

freezing, heating, denaturing

-long term integrity and sterility are also monitored

108
Q

it is important to generate proteins with an N terminus (amino) like found in authentic protein because- (and what is this called)

A

if not, final product may be a mix of several methionyl variants or contain proteins lacking one or more residues from the amino terminus (called amino terminal heterogeneity). NOT desirable because it causes difficulties in purification and characterization of proteins

109
Q

in what direction is DNA extended for PCR, and what does it first require

A

5’ to 3’, needs primer and taq polymerase accomplishes add on

110
Q

3 steps of PCR

A

melt, anneal (cool), extend (DNA synthesis)

111
Q

things required for PCR optimization

A

buffers (KCL and tris usually-can contain others), MgCl2, primer design, cycle number

112
Q

what is the ideal length to sequence for PCR and why, and the ideal temperature and GC content, ideal primer length, acceptable number of runs and repeats

A

18-30 nucleotides, 50-70 celsius, 40-60% GC, less than 5 hairpins. Too short=low specificity, too long= decrease template binding efficacy (higher probability of hairpins, etc), ideal primer length 18-24, 4 bp=acceptable run and repeats (too long increases mis priming and non specific annealing)

113
Q

why is magnesium needed for pCR

A

acts as a cofactor for taq polymerase (needs it to fx), binds DNA and affects primer template interaction (affects interaction with taq too), too much leads to non specific/less stringent binding, too little is a reduced yield

114
Q

what are hairpins

A

formed by intramolecular interaction

115
Q

what are self dimers (homodimers)

A

intermolecular interactions between the two same primers

116
Q

cross dimers (heterodimers)

A

intermolecular interactions between the sense and antisense primers

117
Q

how much lower should the annealing temperature be than the melting temperature

A

5 degrees celsius

118
Q

what is the half life of taq at 95 degrees Celsius and why does this matter

A

-30 minutes: more than 30 cycles of denaturation times of 1 minute, taq is not very effective- therefore ideal cycle number is 25-40. After 30 cycles, won’t see as much yield (plateau effect)

119
Q

the Tm for PCR should not be more than

A

60 degrees

120
Q

how should primers in PCR end

A

3’ in G or C or CG or GC- prevents breathing of ends and increases efficacy of priming

121
Q

what is site directed mutagenesis and what does it need

A

creates a mutation at a defined site- requires a known template sequence; a mismatched oligonucleotide is extended, incorporated into a strand of DNA that can be cloned

122
Q

what is ASO

A

antisense oligonucleotide- short DNA analogue that hybridizes with complimentary mRNA in a sequence specific manner (can result in inhibition of gene expression giving reduced levels of translation of the target transcript)- inhibits ribosomes so they can’t bind and you can’t get expression

123
Q

what are ASOs being used for?

A

researched as potential drugs for cancer and other diseases (“virsen, mersen”) especially linked to dysregulated gene expression

124
Q

what does an effective ASO need to be designed at

A

regions where mRNA is accessible for hybridizations (usually terminal end, internal loops, hairpins, joint sequences, bulges or 10 or more

125
Q

how to deliver ASO (the problem)

A

-unmodified and naked has a net negative charge that can barely penetrate the plasma membrane. Fix with electroporation (by electric field), microinjection, vectors (cationic lipid carriers), covalent conjugation of ASO to a macromolecule (like dendrimer and cell penetrating peptide- CPP4)

126
Q

what does it take to bring a drug to marker?

A

about 1 billion, and 5-10 years

127
Q

what was the first genome sequenced

A

bacteria

128
Q

how many bps does the human genome have

A

3.2 billion (includes all DNA on all chromosomes and genes, intergenic sequences and repeats)

129
Q

eukaryotes can have 5-6 genomes

A

false- 2-3 (nuclear, mitochondrial, plastid)

130
Q

what makes something druggable

A

if the drug can bind to that target. May or may not involve disease therefore separate into non disease and gene disease

131
Q

what are me too drugs

A

another drug already doing jib, new drug works too

132
Q

what is the reason for genetic differences in people

A

genetic variation- 2 kinds 1= most common=SNPs- single nucleotide polymorphisms (single base pair positions which different alleles exist), 2= insertion or deltion of one or more nucleotides

133
Q

polymorphism

A

genetic variation observed at a frequency of more than 1% in a population

134
Q

SNPs- usually have no phenotypic effect

A

true- very common, lots in uncoding regions, some are alleles of genes

135
Q

tandem repeat polymorphisms

A

common polymorphism, variable length sequences that are repated in tandem (microsattelites are 1-6 units, mini are 14-100)

136
Q

pharmacogenetics vs pharmacogenomics

A

genetics= variability in drug response determined by single genes, genomics is multiple genes

137
Q

how many bacterial genomes are completely sequenced

A

over 5000

138
Q

targets in bacteria must be two things** main, and a third

A

selective (only present in bacteria not host-potentially cell wall, inhibiting unique enzymes, disrupting bacterial protein synthesis), essential (essential for bacterium so when we knock it out it dies), wide spectrum (present in all subtypes we target)

139
Q

c value paradox

A

complexity doesn’t correlate with genome size

140
Q

N value paradox

A

complexity doesn’t correlate with genome number

141
Q

k value paradox

A

complexity doesn’t correspond with chromosome number

142
Q

if you have increased number of cells you have increased number of

A

genes

143
Q

karyotyping

A

gene matching in which you compare chromosomes

144
Q

why is the rate/mouse model of choice for pre clinical drug discoveries?

A

mouse and human synteny; humans chromosomes can be cut into about 150 pieces and shuffled to reasonable approximation of mouse

145
Q

why do comparative genomics

A

tells us what are common and unique between species at genome level, genome comparison is the surest and most reliable way to ID genes and predict fxs and interactions, fxs of human genes and other DNA regions can be revealed by studying their counterparts in lower organisms

146
Q

how many bp in the human genome

A

3 billion

147
Q

how many human genes are shared with primates? fruit fly?

A

over 98%, about 60%

148
Q

what is the CGAP and what have scientists learned using it

A

Cancer gene anatomy project, in cancer cells certain genes are damaged or switched off while others are on

149
Q

what are the properties of a database

A

structured, searchable, updateable, cross linked, publicly available

150
Q

2 types of databases at NCBI

A

1) primary- data is submitted by the person who generated it (original submissions by experimentalists, content controlled by submitter) 2) derivative databases- built from primary data (supplement info and create new database)

151
Q

gene annotation and the 2 steps

A

process of attaching biological information to sequences in databases. 2 steps: identify genes on genome, attach biological information to genes and genome.

152
Q

forms of annotations in databases

A

genome annotation, variation mapping, comparative genomics, functional genomics

153
Q

alignment concept (and what is optimal)

A

mutual arrangement of 2 sequences that exhibits where they are similar and where they differ- an optimal alignment is with most correspondences and least differences

154
Q

2 bases for alignment

A

1) structural 2) evolutionary

155
Q

structural alignment

A

when 2 protein sequences have more than 20-30% identical residues aligned, the 3D structure is very similar, and form often follows function (so it implies similar fx as well). Sequence alignment is often an approximate predictor of underlying 3D structural alignment

156
Q

evolutionary alignment

A

similarity is an observable quantity that may be expressed as % identity. Enables to determine if two sequences display sufficient similarity. Evolutionary related

157
Q

what is drugbank

A

a database on drugs, targets and enzymes

158
Q

4 types of chromosome mutation

A

duplication, deletion, translocation, pericentric inversion

159
Q

what does germ ilne gene therapy aim at

A

the introduction of genes into germ cells of omnipotent embryonal cells, human germ cell is not ethical

160
Q

what is somato gene therapy

A

the introduction of a gene into somatic cells

161
Q

what is ex vivo gene therapy

A

cells removed from patient, cultured ex vivo, can be provided with therapeutic gene, the infused or reimplanted back into patient (usually with retroviral vectors)

162
Q

what is in vivo gene therapy

A

organs like lung, brain, heart, etc are not suited for ex vivo, so somatic gene therapy can only be performed by administering gene of interest either locally or systemically

163
Q

where is somatic gene therapy aimed

A

at cells or organ where the disease is manifested

164
Q

how is stable gene transfer accomplished (how does the therapeutic gene integrate into host cell chromosomal DNA)

A

retrovirus or adenovirus mediated gene transfer, or at low efficacy after non viral mediated DNA transfer

165
Q

what is therapeutic gene transmitted to

A

progeny cells

166
Q

what does episomal transfection with and without and organ cause

A

with- stable gene transfer and transmitance to progeny cells

without- only transient infection (episomal DNA is lost upon division)

167
Q

what is the most common approach employed for cancer gene therapy

A

cytokine genes like interferon, interleukins, and GM CSF- they induce a local inflam reaction which destroys both tumor and metastases

168
Q

what is a suicide gene- give an example

A

another way to approach cancer gene therapy, mediate direct cytotoxic or antiproliferative effects on tumor cells and are only effective for localized tumors- phosphorylated ganciclovir is incorportated into DNA of dividing cells, leading to elimination of DNA chain elongation, resulting in cell death

169
Q

what does the bystander effect occur through

A

gap junctions to neighboring cells

170
Q

tumor suppressor genes such as __ are often targeted in cancer, or ___ genes are targeted at oncogenes

A

p53 (which are mutated), antisense are targeted at oncogenes to reduce or abolish their expression. These are for direct cytotoxic or antiproliferative effects and only effect localized tumors

171
Q

a way to target cancer is to protect _____ from toxic effects of chemotherapy by _____

A

hematopoietic stem cells (HSCs) from the toxic effect of chemotherapy by inserting a gene that confers drug resistance (like MDR-1 ie multidrug resistance gene)

172
Q

what does MDR-1 allow patients to do

A

multidrug resistance gene protects hematopoietic stem cells from toxic chemotherapy, so patients are expected to tolerate higher doses of chemotherapy and thus increase effectiveness

173
Q

viral vs non viral gene transfer

A

viral is more efficient, non is naked DNA inserted in liposome and is not as efficient but has no known safety issues

174
Q

methods of non viral gene transfer

A

injection of naked DNA, particle bombardment (gene gun), entrapping DNA into liposomes

175
Q

injection of naked plasmid DNA for non viral gene transfer

A

simplest, reasonable efficacy in muscle and skin, requires a lot of DNA because not that efficient, easy, safe, suitable for large gene constructs, can also be introduced with gene gun (microscopic gold or tungsten pieces forced with it)

176
Q

how liposome encapsulated DNA transfer non viral works; methods to help it

A

DNA is negatively charged so put in positively charged liposome; fuse with membrane of target cell, DNA released into cell. Problem is few that are released into cytoplasm are expressed- fix with adding peptides or virus shells to complex to minimize breakdown of DNA, or attach antibodies for targeting purposes. Safe, efficiencies low

177
Q

how have DNA liposomes and non viral gene transfer been used for CF?

A

CFTR =cystic fibrosis transmembrane conductance regulator gene that codes for protein that transports chloride (CF have defect in this),clinical trials on DNA/liposomes instilled in patients’ noses (same secretory defect as lungs and easier to harvest and measure cells)

178
Q

gene transfer using recombinant viruses (viral vectors) is good because

A

virus has natural capacity to infect cells and deliver genes to nucleus, much more efficient than non viral. Virus is inactivated so can’t reproduce (retro, adeno, adeno associated)

179
Q

if viruses are used to transport genes, must:

A

be replication defective, no undesirable properties, able to accommodate therapeutic gene in its viral genome

180
Q

ideal vector for gene transfer

A

high efficiency (many cells infected or transduced), convenience and reproducability of production, ability to transduce dividing and non dividing cells, ability to integrate into a site specific location in host chromosome or successfully maintained as stable episome, transcriptional unit can be controlled by its regulatory elements, ability to target desired type of cell, no immune response

181
Q

what is a retrovirus and how does it work

A

genome has two copies of single stranded RNA; goes through reverse transcription using RNA as template. Must adhere and enter cell. Uses integrase to insert its reverse transcribed double stranded DNA into human genome and may result in mutagenesis (forms pro virus). Proviral DNA is transcribed and translated into viral proteins which bud from cell wall

182
Q

retrovirus vector properties that make it suitable for gene transfer

A

affect a wide variety of cells with high efficiency, proviral copy stably integrate into chromosomal DNA of cell giving life long correction of target cell type and descendants, sequences for replication can be separated into cis and trans which enables generation of replication defective recombinant retroviruses

183
Q

recombinant retrovirus systems consist of two building blocks

A

retroviral vector (to transfer gene-ie piece of RNA with gene), retrovirus packaging cell (only needed to produce replication defective virus ie the vector)

184
Q

what are retroviruses used predominantly in

A

ex vivo gene transfer; ususally need growth factors to stimulate division of target cells and increase transfer efficiency

185
Q

what was the first gene therapy protocol for

A

adenosine deaminase (ADA) deficiency (lethal inherited disorder leading to severe combined immunodeficiency) (destroys T and B cells because can’t produce uric acid)

186
Q

what are gene marking studies

A

have no therapeutic intent- aim at demonstrating that an exogenous gene can be safely transferred to patient and determine how long this gene is detectable in the patient’s target cells

187
Q

problems with gene therapy

A

short lived (rapidly dividing nature of cells prevents long term correction-need multiple rounds of therapy), immune response, viral vectors (toxic or inflam response or cause disease), multigene disorders (hard to treat- ie heart, BP, alzheimers, arthritis, DB), may induce a tumor (insertion based mutagenesis)

188
Q

how can genetic material of animals be manipulated

A

insertion (transgenes), replacement, deleted (knockout)

189
Q

gene therapy vs transgenic

A

gene therapy- needs a vector and attempts to replace or repair in a developed animal, vs transgenic is grown from a genetically modified embryo from birth

190
Q

what is the most common way to produce transgenic animals

A

DNA microinjection (inject into larger, male pronucleus, prior to first cell division so that all cells will contain the transgene, can be passed on 20-25% of time in detectable levels to offspring)

191
Q

most efficient way to produce transgenic animals

A

in vitro by homologous recombination in embryonic stem cells; derived from mouse blastocyst (can have DNA modified while retaining ability to contribute to somatic and germ cell lines), done by microinjection or transfection then implant into blastocyst and transfer to surrogate animal

192
Q

what happens when you use hematopoietic stem cells vs ES cells

A

ES cells change the whole germ line, hem repopulate a specific somatic cell line(s) (more similar to gene therapy)

193
Q

animals can serve as bioreactors to synthesize recoverable quantities of therapeutically useful proteins, especially in milk (harvested easily mechanically or manually by milking)

A

true

194
Q

gene pharming

A

recombinant genes for desired protein are fused to requlatory sequences of animals’ milk producing genes (targetted exclusively to mammary tissue) (animals not endangered at all)

195
Q

how are yields and price of gene pharming different vs recombinant cell culture

A

they are 10-100x higher, price is 75% less expensive, and can undergo post translational modification

196
Q

advantages of gene pharming

A

protein produced in large quantities in milk, can be harvested mechanically or manually simply by milking, yeileds are 10-100 greater, 75% less expensive, can have post translational modification if required

197
Q

what are knockout mice

A

endogenous gene has been inactivated by replacing it with a null allele (good tool to examine gene function in vivo)

198
Q

what is genetic ablation

A

aka cell ablation or genetic amputation, used to suppress growth of a specified cell line or cell type in an animal rather than suppress gene expression. transgene is under control of gene promoter that is only active in a certain cell population

Biopharmaceutics (3 decks)

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