gene transfer techniques - jagiellonian...
TRANSCRIPT
Prof. Wacław Szybalski McArdle Laboratory for Cancer Research, Wisconsin, Madison,
USA
Birth of gene therapy – 1962 (Elisabeth Szybalski and Waclaw Szybalski)
HPRT+/+ cells
HPRT-/- cells
HAT medium
Gen HPRT
HPRT-/- cells
(HPRT – Hypoxanthine-guanine phosphoribosyltransferase)
H – hypoxanthine A – aminopterin (inhibitor of difolate reductase) T - thymidyne
Genetic transformation Genetic transformation in vitro - modification of cultured cells through introducing the nucleic acids. It can be applied to:
* homogenous cell cultures (both primary and immortalized cell lines)
* cultured tissues
* cultured fragments of organs (e.g. slides of the brain)
* cultured small organs (e.g. fetal lungs)
Culture of murine embryonic lungs
Bovine pulmonary endothelial cells Organotypic culture of retina
Genetic transformation - major methods
- Nucleic acids can be introduced to the cells using:
* physical methods (transfection)
* chemical methods (transfection)
* biological methods (transduction)
History: * first cell cultures (insect cells) - beginning of XX century.
* first established cell lines - 40-ties of XX century.
* first genetic transformation - 1962 by Elizabeth Szybalski and Wacław Szybalski, University of Wisconsin.
Genetic transformation - major methods - despite the method used, the nucleic acid must overcome several barriers:
A. Crossing plasmalemma
* Cell membrane has a negative charge, as DNA or RNA, which blocks attachment of nucleic acids to the surface of membrane.
* Both DNA and RNA are hydrophilic, which blocks the direct fusion with lipid plazmalemma.
B. Release from phagosomes (if nucleic acid is taken by phagocytosis). Can be facilitated by membrane destabilizing compound as chloroquine, viral hemagglutinin A2, artificial fusion protein KALA, GALA)
C. Dissociation of nucleic acid from the chemical carrier (if chemical methods are used)
D. Transportation to the nucleus (can be improved by adding ORI sequence from SV40 virus to the nucleic acid or adding to the chemical vehicle a protein with nuclear localization signal, NLS, e.g. SV40 large T antigen).
Most often, the rate limiting steps are B and D.
DNA delivery pathways with three major barriers: low uptake across the plasma membrane, inadequate release of DNA molecules with limited stability, and lack of nuclear targeting. (A) DNA−complex formation. (B) Uptake. (C) Endocytosis. (D) Escape from endosome. (E) Degradation (edosome). (F) Intracellular release. (G) Degradation (cytosol. (H) Nuclear targeting. (I) Nuclear entry and expression.
Luo & Saltzman, 2000
Genetic transformation - major methods
- The method of gene transfer used
- Type and quality of cells
- Confluence of cell culture and proliferation of cells.
- Size and quality of nucleic acids (most methods work well up to 15 kb DNA; contamination with phenol decrease the efficacy).
Efficacy of gene transfer depends on:
Endothelial cells
Factors influencing efficacy of transfection: - Purity of DNA * removal of contaminating remnants of phenol and bacterial toxins. - Proper conformation of plasmid DNA * the highest efficacy is obtained for plasmid at CCC form (the highest rate of transfer and resistance to endonuclease activity) * linearized plasmid is better for stable transfections (more efficient recombination and induction of ligases' activity by free ends of plasmids)
Efficacy of gene transfer - quality of DNA
- Plasmids remains in nucleoplasm in an episomal form and do not integrate to chromosomes. - In most cell lines they are present in the nucleus for 12-48 h, and then are gradually degraded. - In some cells (e.g. human embryonic kidney cells HEK 293) plasmids can function at the same level even for 2-4 days.
Fates of DNA within the cell
HEK 293
- The most often used plasmids do not replicate in the eukaryotic cells. - Some plasmids contain sequences, which enable replication in the permissive cells. There can be: * replicon from viruses of family Papovaviridae. * sequence from simian virus SV40 (the most often used) - in monkey cells (rhesus or green african monkey) * sequence from mouse polyomavirus (MPV) - in murine cells * sequence from bovine papilloma virus (BPV) - in bovine and murine cells * sequence from human polymoma BK virus (BKV) - in human cells - These sequence can be not sufficient to allow proliferation. For example for proper work of SV40 replicon the presence of SV40 large T antigens is necessary. Thus: * the plasmid should contain the sequence encoding large antigen T sequence * experiments should be performed in COS cell lines (green african monkey fibroblasts), containing integrated genome of SV40 virus. In this case, under optimal condition even 100 000 copies of plasmids per cell can be obtained.
Self-replicative plasmids
- In most cases plasmid DNA exists as an episomal element and is gradually degraded. Additionally it is lost during the replication of cells, thus the percentage of transduced cells gradually decreases. Therefore the effects of transfection are short-term.
Transient and stable transfection Transient transfection
- Some vectors - especially retroviral vectors - introduce the transgene to the host genome. Such DNA replicates together with the rest of chromosome and is traded to the all doughter cells. Therefore, the effects of transfection are long-term.
- Integration can occur also for other vectors e.g. plasmids or adenoviral ones, but this process is very uneffective ranging from 1 per 100 to 1 to 1000 successfuly transduced cells.
- Transgene usually builds into more-less random site of genome. - Site-specific integration using a homologous recombination mechanism is possible,
but more diffucult and rarely used.
Stable transfection
Transient and stable transfection
Selection of stably transduced cells
Vectors for a stable transfection should contain a gene for selection of transfected cells, such as:
- antibiotic resistance gene (the most often selection): * neomycin * puromycin * bleomycin * zeomycin - GFP or RFP which can be used for sorting Usually cells for 1-2 days after transformation are cultured in the routine medium,
which is later on replaced with the selective medium for 2-4 weeks. There is possible to co-transfect cells with a target plasmid and second plasmid
containing antibiotic resistance gene (at molar ratio 10:1).
Geneticin
Transient and stable transfection Selection of stably transduced cells
Vectors for a stable transfection should contain a gene for selection of transfected cells, such as:
- gene coding for protein necessary for survival of cells in which the endogenous gene encoding this protein is mutated and inactive. Such cells can survive only in a special supplemented media. They can survive in routine media only after successful transformation.
* chinese hamster ovary cells without dihydrofolate reductase (DHFR), coding for enzyme converting dihydrofolate to tetrahydrofolate. Such cells can be cultured only in media supplemented in nucleozides. After transformation with vector containing DHFR gene, they can survive in routine medium.
* cells with inactive thymidine kinase (Tk), necessary for synthesis of deoxythymidine monophosphate (dTMP) from thymidyne. In normal conditions Tk activity is not necessary, as dTMP may be synthetised de novo from carbamoilophosphate. However, in the presence of the inhibitor of this pathway (aminopterin), dTMP must be synthetised by Tk. Thus, media with aminopterin can be used for selection of successfully transduced cells.
Expression of transgene does not depend only on the promoter and other regulating gene sequences, but also on: - region of integration (possibly the most important) - number of copies integrated (not direct relation): 1-10 after electroporation up to 100 after calcium phosphate transfection. - quality of transgene (e.g. rearangements, mutations) - availability of transcription factors, polymerase and translation initiation factors - production of untypical RNA, which can inhibit transcription - inhibition of transgene expression as an effect of promoter methylation - selection and overgrowth of cells without active transgene
Expression of transgene in stably transfected cells
Incorporation of transgene into chromosome can cause: - changes in the level of expression of endogenous genes - very often transgene integrates into transcriptionally active regions
- insertional mutagenesis - insertion within the gene, deletions of gene fragments
- induction of endogenous gene expression from a strong transgene promoter (especially from retroviral LTR sequences)
- changes in methylation pattern, even in fragments distant from transgene
Unexpected effects of stable transfection
control transformed
- Immortalization of cell lines (transformation with use of e.g. retroviral vectors) by introduction of gene encoding: * large T antigen of SV40 virus * telomerase * adenoviral gene E1a * genes E6 and E7 of human papilloma virus (HPV) * genome of Epstein-Barr virus (EBV), - Immortalization is not a cancer transformation - cells may have a contact inhibition.
Aims of in vitro transformation
immortalized endothelial cells
cancer cells
Aims of in vitro transformation
- Studies on function of genes:
* Introduction of expression vector to obtain high level of transgene expression and analyse the effects. Expression can be governed by constitutive promoter or regulable promoters. Regulation can be obtained e.g. by hypoxia or some antibiotics.
* Introduction of siRNA to inhibit activity of the gene studied and analyse the effect. The siRNA can be synthethised and introduced by means of chemical carriers or can be produced in the cell after transformation with vector coding for siRNA.
Note: in this type of experiments one must use a control vector to recognize the effect of transgene or siRNA from effects of procedure itself. Controls:
* For overexpression of transgene: empty vector or vector containing control gene
* For siRNA: scrambled RNA
Aims of in vitro transformation - Studies on regulatory regions. Cells are transfected with vectors containing a reporter gene, whose expression depends on the regulating sequence studied.
- Reporter genes should: * encode protein, whose expression is easy for detection and measuring, preferentially with sensitive colorimetric or luminometric methods * differ from endogenous genes to improve specificity. * regulated at the expression level * not require post-translational modifications modulating its activity or stability
- Examples of reporter genes: * chloramphenicol acetyltransferase (CAT) * β-galactosidase * luciferase * Green Fluorescent Protein (GFP), * Red Fluorescent Protein (RFP), * soluble alkalic phosphatase (SEAP) * β-glucoronidase (GUS).
β-galactosidase staining
Regulatory sequences studies with reporter gene assays - Reporter genes can be used to study activity and specificity of: * Promoters (full length or deletion constructs) * Transcription factors * Polyadenylation signals
- Constructs are introduced to:
* different cell types
* organotypic cultures
- Distinct levels of expression of reporter genes reflect the tissue-specific differences in acivity of regulatory sequences.
- Because level of expression can be strongly influenced by transfection efficacy, the control plasmids with reporter gene driven by constitutive promoter must be always used.
Cell-type specificity of regulatory sequences
heart development (cardiac troponin-T)
- Production of recombinant proteins: * transgen should be driven by a strong promoter. * produced proteins can be labeled with tags such as His lub c-Myc, - Tags should: * be small (8-10 amino acids) to reduce influence on the protein functions * allow for detection of proteins using universal antibodies
Aims of in vitro transformation
- Studies on cellular localization of proteins * Cells are transfected with constructs coding for a fusion molecule consisted of protein studied and fragment of GFP or RFP. * Localization of these proteins in the living cells may be checked under fluorescent microscope * Technique can be used for studying translocation of proteins within the cells, e.g. kinases or transcription factors
Aims of in vitro transformation
cellular retinoic acid binding protein fused with GFP
- Labeling cellular compartments: * Cells are transfected with constructs encoding proteins typical for given organellum, fused with different variants of GFP. * Technique can be used for studies of structure of cytoskeletone or localizations of mitochondria
Aims of in vitro transformation
- Studies on protein-protein interaction:
* FRET ( fluorescence resonance energy transfer).
* BRET (bioluminescence resonance energy transfer).
Aims of in vitro transformation
- Screening for potential drugs. * Transfection of cells with an expression vector coding for a target protein * Transfection of cells with a reporter vector to measure activity of the target promoters or target transcription factors. - Transfections are performed in 96- or 384-well plates (ang. cell based high-throughput assays) - Test should be simple, cheap, fast and reproducible.
Aims of in vitro transformation
TATATA PPRE
pio
TATATA PPRE REPORTER
REPORTER
pio
pio
pio
pio
pio
pSV40 human PPARγ
- Production of viral vectors for experiments and clinical trials. * Transiently transformed packaging cells producing in trans lacking viral proteins (e.g. for producing retroviral vectors or AAV vectors). * Stably transformed packaging cells producing in trans lacking viral proteins (e.g. for producing retroviral vectors or adenoviral vectors).
Aims of in vitro transformation
RESULTS:
1,00E+00
1,00E+03
1,00E+06
1,00E+09
1,00E+12
1,00E+15
1' 2' 3' 4' 5' 6'
passages
titer
[pfu
/ml]
Aims of in vitro transformation
- Production of viral-like particles (VLP) - empty viral capsids, which are strongly immunogenic and can be used as a vaccine. * production in yeast * production in insect cells
HPV - VLP Rotavirus - VLP
Transformation of mammalian cells - Major strategies of gene transfer:
* Physical methods - introduction of DNA to cytoplasm or nucleus by inducing of local, temporary and reversible damage of plasmalemma. Special equipment is usualy required.
* Biochemical methods - introduction of DNA using the chemical carriers (usually polycationic lipids or polyamines), which neutralize negative charge nucleic acid and facilitate phagocytosis or fusion with membrane. Routine cell culture equipment is required.
* Biological methods - introduction of transgene using viral vectors. Sometimes also bacteria or eukaryotic cells are used. Biohazard cell culture equipment is required.
- Gene transfer techniques should be:
* effective
* reproducible
* not toxic
* easy, fast and affordable
Physical methods
- Electroporation:
* Cell cultures are exposed to short electric impuls (100- 1500V; several ms).
* Effectivity depends on: time, voltage, temperature, amount and conformation of DNA, presence of ions.
* Very effective, but very toxic.
Physical methods
- Transfection with ultrasounds:
* Similar to electroporation, but cells are exposed to ultrasounds.
* Can be effective but is toxic.
* Can lead to damages of DNA.
plasmid injection plasmid injection +
ultrasounds plasmid injection +
ultrasounds
injection to skeletal muscle
- Photoporation:
* Exposure of cells to laser light to induce formation of pores in plasmalemma.
* Can be used for a small number of cells.
* High proportion of stable transfection.
Physical methods
Physical methods
- Microinjection:
* Injection of DNA directly to the cells.
* Requires skillful researcher :)
* Very small amounts of DNA are needed.
* Efficacy of integration with chromosome - up to 20%.
* Used for production of transgenic animals.
* Can be used for introduction of BAC
Physical methods - Gene gun:
* Constructed in 1987.
* DNA molecules cover particles of gold or wolfram and are shot to the cells or tissues with help of high pressure of helium.
* Efficacy depends on type of the gene gun and type of tissue
* Used mostly for production of transgenic plants
Dr. John Sanford, inventor of gene gun
gene gun at work
Biochemical methods
- Calcium phosphate
* First method of gene transfer (1962), still in use
* Effective in some cell lines, but not in others
* Can be toxic and can induce differentiation of some cell types.
* Recommended for introduction of several plasmids.
CHO cells covered with precipitates of different size
Biochemical methods
- Lipoplexes:
* cationic lipids
* anonic lipids
* neutral lipids
- Features of lipoplexes:
* high efficacy in some cell types and some efficacy in others
* reproducible results
* can be used for introduction of different nucleic acid (from siRNA to YAC)
* must be optimized for each cell line (proportion of DNA : vehicle, amount of DNA, times).
Biochemical methods
- Polycationic lipids
* facilitate phagocytosis or - rarely - fusion
* can protect against nucleases
* often mixed with neutral lipids (e.g. DOPE)
* expensive but effective - very often used
- Examples:
* Lipofectamine
* FuGene
* Maxifectin
Biochemical methods
- Dextran DEAE (positively charged polyhydrocarbonate)
* one of the first vehicles for gene transfer
* facilitates phagocytosis of DNA
* relatively cheap but rarely used
* effective in some cell lines (but not in primary cell cultures), can also increase efficacy of retroviral vectors
* can be strongly toxic to cells
Biochemical methods
- Dendrimers - polymers composed of polyamidoamids with very complex, multilayer structure, with initiation center (NH4+ or ethylenediamine) and regular, dendritic branches. They are positively charged because of many amine residues.
* water-soluble, easy for handling
* facilitate phagocytosis
* can facilitate release of DNA from phagosomes and protect DNA from nucleases
* not toxic for cells
* do not activate cells - preferentially used for studies on function of genes
SuperFect
Biochemical methods
- Cationic peptides
* not effective and relatively rarely used
* facilitate phagocytosis of DNA or fusion of complexes with membrane
- Examples:
* poly-L-lysine (not efficient)
* poly-L-ornithine
* poly-L-arginine (fusigenic peptide)
poly-L-lysine
Biochemical methods - Spermine - natural polyamine, one of the simplest linear polymer, with many amine residues
* facilitates phagocytosis
* Rarely used as a single agent, but added to other vehicles
- Polyethylenamine - polycarbohydrogene, containing nitrogene
* facilitates phagocytosis
* helps to destroy endosome and release DNA to cytoplasm and improves transportation to nucleus - used as a supplement to other vehicles
- Polybrene
* improves transduction by retroviruses
* can be effective only in some cell lines
* rarely used Polybrene
Biological methods - Retroviral vectors (based on RNA viruses):
* one of the most often used vectors
* introduce transgene into host genome - are perfect for developing stably transfected cell lines
* do not interfere with cell functions
* can infect dividing cells only (with exception of lentivirus)
* capacity: ~7 kb
gag pol env
transgene
LTR
LTR LTR
LTR
retrovirus
retroviral vector
Application of retroviral vectors in the clinic - Modification of cells with retroviral vectors for gene therapy (retroviral vectors cannot be used for transduction in vivo, as they are immediately destroyed by complement cascade) * transduction of mature cells * transduction of progenitor cells
liver
LDL-R
LDL-R- LDL-R+
LDL-R+
Biological methods
- Adenoviral vectors (based on DNA viruses):
* one of most often used, universal vectors, which can be produced at high titers
* very efficiently transduce many different cell lines, both proliferating and not proliferating
* remain as episomal element, giving very strong but short-term expression
* capacity: ~9 kb
* can be used for introduction of very long DNA (e.g. BAC) - in this case DNA is condenced and attached to the outer part of capsid and transported to cytoplasm together with vector.
E1
ITR ψ+ ITR
transgen ITR ψ+ ITR
adenovirus
adenoviral vector
Suzuki et al. 1998. FEBS Lett 425: 436-440, Soudais et al. 2000. J Virol 74: 10639-10649.
Comparison of efficacy of plasmid and adenoviral vectors for delivery of transgene to murine liver
Naked plasmid Adenoviral vector
Biological methods - AAV vectors (based on DNA viruses, adeno-associated viruses):
* transduce many different cell lines, both proliferating and not proliferating
* especially useful for gene transfer in vivo, as they do not induce strong inflammatory response; can sustain relatively long-term expression
* remain as episomal particles (despite capability of wild-type AAV to intergrate in a strictly defined site on chromosome 19).
* capacity: usually ~4.5 kb
AAV
AAV vector
In most glioblastoma, human telomerase reverse transcriptase (hTERT) is reactivated in the cell, maintaining the telomere portions of the genome. The 27kDa C-terminal polypeptide, when overexpressed, can cause a dysfunctional hTERT, which can induce senescence. Ng et al. Nature 2007
AAV vectors
Biological methods - Hepatoviral vectors (based on EBV and HSV):
* can transduce neurons (HSV) and lymphocytes B (EBV), but can be used for other cell lines
* very high capacity (30-40 kb)
* in many cells they can replicate, giving episomal, but stable expression of transgene
Biological methods - Poxviral vectors (based on Vaccinia virus):
* can transduce many cell types, both proliferating and not proliferating
* give transient but very strong expression of transgene
* rarely used, but may be eployed for transfer of very long fragment of DNA
* remain as episomal particles
Biological methods - Alphaviral vectors (based on Semliki Forest or Sindbis virus):
* can transduce many cell types of vertebrates and invertebrates, both proliferating and not proliferating
* can be produced at high titers
* give transient but very strong expression of transgene; thus they are preferentially used for production of recombinant proteins for structural studies
* less suitable for analyses of gene functions, as transduction inhibits production of many endogenous proteins and can be toxic to cells
* do not produce DNA, thus they cannot be used for stable transduction
Next lecture – 8th May
When exam? 5 or 12th June (before session)
19 or 26th June
Only one term!