[ ]Summarised study of 10 technologies that could be used for Gene Therapy, in this assignment the disease in study is ‘Menkes Disease’ related to poor assimilation and absorption of Copper in the body due to mutation or mal-functioning of the ATP7A gene (truncated form is produced).

2011

Pranamee Sarma psarma@hawk.iit.edu

1) LASER CAPTURE MICRODISSECTION TECHNIQUE

ADVANTAGE DISADVANTAGEInvolves no manual dissection Complex setup, such as computer programs, laser

etcFull-proof cell selection and transfer to tubes Targetting large number of cells is tediousTissue retains its morphology, only the specific portion is cut

Some times cells are scattered rather than localised so becomes a problem in fine tuning the target

Pure population of cells Full time attention required, process is quite long

Title : Isolation of the seminiferous tubules (containing mature spermatids) from the testicular tissue (present in the outer edge of this tissue) of a)Menkes affected male b) Normal male to be able to isolate the DNA content of the germ cell.

Introduction : Laser Capture Microdissection (LCM) is a low beam laser-assisted method to isolate or ‘dissect’ pure and micro-portions of specific cells from a clustered or diverse tissue/cellular system. The best part being that we can select the specific part on the computer screen connected to the LCM

Working : The laser beam melts the thermo-labile film attached to stick to the target cells. We get the final cells in eppendorf tubes stored beneath the cutting area of the system to collect the sample (there are other ways to select the cells as well). Once the area is selected, a tissue capturing eppendorf tube-cap is placed on the targetted area and a low powered infra-red beam laser cuts the target area, the target cells present on the cap are then transferred to the tube. The tubes placed underneath (as mentioned earlier) have the target tissue cells which can then be taken for further studies. The main catch of this experiment is ‘tissue-area selection’,’laser beam targetting’ and ‘computer monitoring of the process’. A loss at one of them will produce unsuccessful results.

COST- Approximately 1, 43, 640$ (includes the microscope, computer software and hardware)

Application (Gene therapy, e.g Menkes Diesease)

STEP 1 : Testes tissues from both source (normal and diseased); observe under microscope where we see the outer edge with spermatids and immature spermatozoa (seminiferous tubules)

STEP 2 : We use LCM to selectively cut/dissect the outer edge cells from the rest of the tissue by marking the specific regions to be cut, on the computer screen. Laser beam targetted to these areas cuts and creates visible holes in the original tissue.

STEP 3 : The cells selected are collected on the caps of the eppendorfs used. The seminiferous tubules /sertoli cells are distinguished and isolated for further DNA isolation(also RNA) in our case (Centrifugation-sperm pellet-extraction buffer and proteinase K-centrifugation-pellet-DNA; add dye or buffer accordingly). We can run the DNA obtained on Agarose gel electrophoresis and observe the band differences (based on dye) and then we can perform DNA profiling or fingerprinting to study the differences in the normal and diseased ( the disease in our case is X chromosome-linked recessive disease)

2) REVERSE TRANSCRIPTASE – PCR (RT-PCR) TECHNIQUE

ADVANTAGE DISADVANTAGEEarly diagnosis of diseases Need of special primers, thus tediousMeasuring gene expression levels Can pick up contaminants from the

surroundingcDNA production, no introns, thus helpful for Use of two different enzymes, makes it a little

Title : mRNA from diseased and normal organism can be taken and ‘Reverse transcribed’ to its DNA complement, cDNA for further studies such as gene therapy.

Introduction : It is a variant form of normal PCR (Polymerase Chain reaction) which is used to ‘polymerise’ or amplify the required sample of nucleic acids. In case of RT-PCR, there’s an additional step of ‘Reverse transcription’ in the beginning where, enzyme ‘reverse transcriptase’ transcribes RNA back to DNA and thus the cDNA is amplified.

Working : The amplification of cDNA occurs in the ‘traditional’ number of 3 steps; FIRST : (the main difference than PCR) Reverse transcription of mRNA to DNA (ds) using reverse transcriptase at 50⁰C; SECOND : Double stranded (ds) DNA melt down and inserted primers anneal along the single stranded DNA at 92⁰-95⁰; followed by primer annealing at 65⁰C; THIRD: A special (high temperature) enzyme Taq (DNA) polymerase extends DNA using the primers therefore leading to cDNA amplification.

Requirements : Thermal cycler, Computer, data and collection equipments, mRNA, PCR special tubes, enzymes

COST : 40, 000$ - 50, 000$

Applications (for gene therapy, e.g in Menkes Disease):

STEP 1 : mRNA is isolated from the technique used above (sperm cell with LCM) using oligodT beads (pulls out polyA tail and thus mRNA).

STEP 2 : In 2 tubes, the mRNA of normal male and Diseased male are treated at a specific temperature with Reverse transcriptase enzyme to give DNA (cDNA) ds copy.

*Through this process we can detect any mutations (e.g Exon skipping or splicing abnormalities, that are most common in Menkes) by comparing the cDNA copy of normal to diseased from the mRNA that we had and thus locate the sites of mutation.

STEP 3 : Then the targetted cDNA is amplified DNA sequencing (Automated DNA sequencing) with radiolabelled nucleotides from both the samples and then by comparison we can clearly notice the mutation that has occurred (mismatch or nonsense mutation)

genetic engineering and modifications between prokaryotic genes and eukaryotic genes

complicated to handle compared to traditional PCR method.

3) QUANTITATIVE POLYMERASE CHAIN REACTION

Title : qPCR for the above sample is necessary for quantification and amplification of the cDNA produced

Introduction : It is a PCR (Polymerase Chain reaction) which is used to amplify and quantify the required sample of nucleic acids(cDNA in this case). In case ofqPCR, flourescent dyes (DNA probes) are used to measure/quantify the amount of DNA(e.g) present in the starting sample and the final sample. It is basically amplification detected by flourescence.

Working : The amplification of DNA occurs in 3 steps; FIRST : Denaturation, at 95⁰C; SECOND : Annealing, at 65⁰C, double stranded (ds) DNA melt down and synthesized primers anneal along the single stranded DNA ; THIRD: A special (high temperature) enzyme Taq (DNA) polymerase extends DNA at 75⁰C using the primers, leading to cDNA amplification.

These temperature important as they are crucial for Primer elongation and DNA hybridisation.

COST : 50, 000$ or more depending on the probes used.

Applications (for Gene therapy e.g Menkes Disease)

STEP 1 : We use the previous technique’s sample for quantification. RT-PCR is coupled with qPCR for better results.

STEP 2 : We label the cDNA from RT-PCR with flourescent probes so that through qPCR we increase the specificity for our samples to enable proper target cDNA quantification for Menkes Disease treatment and analysis.

STEP 3 : The primers used in the Annealing step are Probed and thus anneal with the specific cDNA product enabling flourescence activity to be observed through the plotting of a graph between Flourescence activity against the number of thermal cycles of PCR. Increase in the activity will show an increase in the number of target cDNA Copies (quantification).

*We can also use this technique to quantify GENE EXPRESSION when we modify the Promoter sequence for the ATP7A gene of Menkes Disease to observe the pros and cons of this therapy (promoter modification)

ADVANTAGES DISADVANTAGESQuantifying target sample with precision and specificity; no primer dimers

Costlier than most of the PCR variants due to use of specific probes and extra equipments

Better method to measure Gene expression levels

Complex with high techincality involved

Reliable method of sample/DNA detection Larger set-up, larger work area

4) DNA Gel Mobility Shift Assay and DNA Footprinting

ADVANTAGES DISADVANTAGESPrecise method to study interactions Special conditions need to be maintained for

high efficiencyReveal protein and nucleic acid interaction Not all protein-nucleic acid interactions are

Title : Gel mobility shift assay is to study the affinity reactions between protein and DNA or RNA using DNA footpringting in conjunction

Introduction : The concept of both the techniques is similar in the fact that both observe DNA fragments bound with proteins or no proteins. The difference being, Gel mobility shift assay studies the shifting of a band (either up or down) on binding to a protein which can be observed comparing to data from DNA Footprinting.

Working : DNA for study is run in different lanes, with and without binding proteins. In Gel mobility shift assay, we will observe that the DNA bound with protein (heavy) has shifted ‘up’ in the position in lane compared to the same DNA fragment which has no protein bound (therefore, down). In the DNA footprinting assay, in similar sample we add Dnase I which cleaves protein unbound DNA at random sites and leaves the protein-bound DNA intact. The gel run with these samples, can be autoradigraphed (using DNA probes in the samples) and the difference in a conitnuous cleaved Lane and protein-bound DNA protected lane can be observed by the dissimilar patterns and a missing portion of DNA (GAP, due to proteins bound).

COST : Electrophoresis – 1600$ approx; General accessories – 500$, Chemicals- 100$

Applications (Gene therapy, e.g. Menkes Disease) :

STEP 1: We can test the promoter mutation that we plan to do for Menkes diesease (as mentioned in the previous page) by this method combination.

STEP 2: We can mutate the promoter sequence(original promoter position can also be determined by this process; RNA polymerase binding to promoter region, -10 and -35 sites). Once the promoter is mutated we can use this methodology to see if the RNA polymerase along with the other Transcirptional proteins bind to the the promoter site (new). The promoter sequence, if in the proper site (-10 and -35), will be bound by the RNA polymerase and a gel run with its fragments, will show a gap in this region compared to original fragment electrophoresed. To compare the results we will have to perform both DNA Footprinting (for the new promoter sequence) along with the Gel mobility shift assay (for the RNA polymerase + Proteins bound to the new promoter).

STEP 3: If successful, we can use this process in up regulating theATP7A gene transcription for treatment (gene therapy) of Menkes Disease

complexes solvedLow cost. Technically tedious

5) SDS-GEL and 2D GEL ELECTROPHORESIS

ADVANTAGES DISADVANTAGESConvenient visual method of molecule detection Agarose gels are temperature sensitive, esp. Heat

Title : Gel electrophoresis of biomolecules to separate them on the basis of size/charge where network of agarose molecules(gel) serve as a sieving medium

Introduction : Biomolecules such as Proteins and nucleic acids have to be separated which is done by Electrophoresis of the sample (with a dye/buffer) along a gradient (pre-decided) on an agarose gel with different conditions. In case of SDS-Gel (PAGE;separates proteins), a special component called Sodium Dodecyl Sulfate (SDS) is added to the separating gel part while preparation. In case of 2D Gel electrophoresis, the separation if by 2 directions (1 D) and then the other direction is perpendicular to the previous run.

Working : In the SDS-PAGE technique the main catch is addition of SDS ( a detergent) in an optimum amount so that the SDS denatures the protein molecules making it negative (constant charge for all), thus final separation is on the basis of Mass. In the 2D Gel electrophoresis, the biomolecules, e.g Proteins are first separated on 1D i.e on one property. The 2D continues from there for separation on a second property. The basic principle for this system lies on the fact that no two protein molecules (in this case) will be similar in 2 properties. So if not separated through 1D they are bound to do so in 2D

COST : Chamber and kit = 1600$ approx; Accessories and chemicals = 600$ approx

Applications (gene therapy, e.g Menkes Disease) :

STEP 1 : We got proteins from both of the normal and diseased Males. We need to test them for their properties with the original protein (ATP7A gene). We take one sample of protein from each and run in a gel in different lanes. E.g.

LANE 1 2 3 4 5

STEP 2: We can thus combine 1D as SDS and 2D for a different property and get a result similar to above. E.g as we can se, protein from normal male gives us two bands compared to the molecular weight of the protein marker band, a similar band is seen with the ATP7A from promoter mutant 1, but not from the diseased or the ATP7A from promoter mutant 2 form. Thus for therapy then we can use the promoter mutant 1 form.

*We can thus validate our therapy with such comparisons.

Better resolution than the rest, due to dyes Some dyes (EtBr, e.g) are harmful for humansFast process ( 1-2 hour run) Quantification of molecule is approximateEasy to set up Hard time preparing solutions with exact

concentration6) Cell Culture System

ADVANTAGES DISADVANTAGESProduction of human vaccines and drugs Contamination problems of cell linesHelpful in Gene therapy Tedious maintenance of cell linesGrowing of organisms, such as plants, in vitro Chances of contact inhibition in the system for

Title : Culturing or growing of cells isolated by LCM/etc under special conditions and medium to give a cell mass.

Introduction : We isolate cells to study, but for this they need to be maintained in vitro with conditions suitable for their growth and somewhat similar to their in vivo conditions. The cells grown into a mass are then referred to as a Cell culture system e.g Primary cells or secondary cells. These can then be stored and preserved (immortal cell lines) for study in the future, especiall for human diseases and benefit.

Working : Cell cultures can be prepared by creating special in vitro conditions with various machines, chemicals and technologies. A cell is isolated from a specific part of an organism (Study organism) and then grown on a nutrient medium, placed under an incubator (growth chamber,etc) for a specific period of time. The cell culture obtained directly from the first isolation and cell growth is called Primary cell Line. A Secondary cell Line is produced from a cell culture grown from cells taken from the Primary Cell Line. The secondary cell line can be maintained for a longer duration compared to the Primary line.

COST : Glasswares cost 200$ approx; Equipments and lab set up approx 100, 000$

Applications (gene therapy; e.g Menkes Disease; Sperm cell isolated using LCM technique) :

STEP 1 : Spermatozoa from the sertoli cells of testes can be grown as cell culture. From the primary cell line, cells taken can be injected with the new gene or new enhanced promoter mutant screened from above techniques into the cell. These cells can then be cultured on medium such as those for Embyronic stem cells (closest match), etc with addition of serum proteins and stimulating hormones.

STEP 2 : Temperature is maintained at the body limit of 37⁰C in the growth incubator and cell kept in the medium for a time period.

STEP 3 : The growth media should be maintained and refreshed time to time for a better productivity. The cells can be either grown on a small petri dish or a large flask.

*We can observe all the changes and the manipulations in this system itself, which mimics in vivo conditions in the laboratory.

cells growingExtensively used in protein study and production High cost

7) Western Blot Technique

ADVANTAGES DISADVANTAGESEasy method to detect Protein amount and expression

Complicated set-up

Helps in detecting immunogenic reactions Cost factor arises due to X-ray filming and

Title : To determine the size of a protein or quantify protein expression

Introduction : Western Blot technique is a small diversion from the usual Southern and Northern blot techniques. It basicall works on the ligand-substrate binding wherein specifically protein binding to antibodies or etc, is made use of. The technique uses both gel electrophoresis (normally, SDS-PAGE) and Western blotting (basically transferring the gel-run bands onto a Nitrocellulose membrane)

Working : The SDS-PAGE seperates the proteins on the basis of their molecular weight. This seperation is also based on the concentration of the acrylamide gel. The gel thus obtained is then ‘blotted’ onto a membrane (nitrocellulose), transferred from cathode to anode. Once the proteins (bands)are transferred from gel to the membrane, the membrane is then probed with target protein-specific antibodies which have the property of (chemical reaction, colorimetric) chemiluminescence based on the activity of antibody-protein binding detected by an detector which records this activity and thus aids in quantifying protein activity.

COST : Kit (containing buffers, antibodies, enzyme conjugates costs 300$. Electrophoresis process – 2000$ approx, Membranes – (15 sheets) 107$, Detection kits – 323$, X-Ray film- 70$

Applications (gene therapy, e.g Menkes Disease) :

STEP 1 : We can use this method to detect our new-promoter-induced proteins and their interaction with the standard antibodies (in ase of normal). Solubilise the proteins (normal and dieseased) in a suitable buffer and prepare for SDS-PAGE. Once the gel, as obtained above, is produced, we stack it over a pile of filter papers, with a nitrocellulose membrane in between and kept in a buffer tray. (Cathode and Anode transfer method can also be used).

STEP 2 : The induced and uninduced (gene) proteins bands are transferred onto the membrane in the same pattern as in the gel. This membrane is then treated with buffer, antibody solution, washed again for free antibodies. The antibody specific to the target protein binds to the band area with that protein (normal and induced) and in turn produces a chemical luminiscence which can be detected. The membrane is exposed to X-ray film for about 1 hour, in a dark room. Processed and visualised in the dark room, showing luminescence (bright ; white areas) where the antibody is bound to the protein. This way we can validate our new modified protein (ATP7A) which is almost similar to the normal proteins (antibody binding property).

different kits that are usedHigh level of sensitivity and precision Needs extremely careful handwork while

transferring of bands from gel to membraneDetects even minute protein activities Time consuming method

8) Mass Spectrometry for Protein Analysis

ADVANTAGES DISADVANTAGESInsoluble samples can also be used Volatilisation of samples is a tough step

Title : To characterize and identify proteins using Mass Spectrometry (MS)

Introduction : The MS identifies the characteristics of the proteins (broken into peptides) which can then be studied comparing to established protein databases. They basically ionise the molecule of study (protein) so that these can be manipulated under the magnetic fields of the system to determine the m/z(mass/charge) ratio. (* Magnetic fields can only workd on charged ions; cations in this case).

Working : The MS is basically divided into three parts -1) Ion Source (ESI, Electrospray ionisation, or MALDI, Matrix-associated laser desorption or ionisation, for proteins); 2) Mass Analyzer; 3) Detector. The protein sample is prepared for the first step (ionisation). The sample is hit by electrons giving rise to a radical (cation) by losing electrons. The charge of this ion is usually 1, thus m/z ratio generally helps us determine the ‘Mass’. During the process, the original sample gets fragmented due the peptidase activites. The Mass analyzer basically focusses different ions of different mass (fragments) onto the detector, directed by the magnetic field. When an ion hits the detector screen, a signal is recorded which is then plotted in a graph through the computer. This is compared to the established database of the sequence of the protein and related fragment sequence is then determined. This also gives way to the molecular structure of the protein.

COST : 80,000$ - 100,000$ (depends on the type)

Applications (gene therapy, e.g. Menkes disease) :

STEP 1 : We have protein samples from normal and diseased males. We can use MS to determine the characteristics of there two different protein (ATP7A) samples to mark the difference and also to understand their fragment organisation plus the sequences.

STEP 2 : We take the samples one by one, not together to avoid complexity in result interpretations.

STEP 3 : For instance Protein N (from normal) is ionised into N⁺ (z=1), the graph observed will have the first peak showing N⁺ (original) and then subsequently the fragments of N would show up which can then be compared with the database and the Diseased sample. Protein D (from diseased) will be similarly ionised into D⁺ and then graph with D⁺ as the first peak (molecular weight can be determined by this).

STEP 4 : We can compare the data from both and then design a scheme for a genetically modified protein, with natural properties (including the structure).

*Through this method we can also determine

- ATP7A protein interactions - ATP7A pre-mature and mature form; These will further help us in our gene therapy.

Non-polar samples can also be used Thus, difficult with non-volatile compoundsProtein characterisation, mass and structure detection is possible

Diffuculte to clean due to set up, therefore chances of contamination possible

Small sample size is also good High maintenance costs 9) DNA Microarray (Chip technology in biology)

ADVANTAGES DISADVANTAGESLots of information in single go Highly Attentive skills requiredWe can study gene typing in one experiment Micro arrays need to be fabricated everytime

according to the needs of the experimentSmall chip, therefore no storage problems Might have hybridization problems

Title : To study and detect different levels of gene expression in organisms using microarray (chip) system

Introduction : DNA Microarray is a small chip with organised rows of specified size allowing DNA (cDNA) to hybridise and a scanner to detect the activity of subsequent gene profiling experiments on the chip. It is a convenient method observe gene expressions, genotypes, mutant genotypes.

Working : The Microarray is basically a solid chip, with attachement fabrications for cDNA. We say cDNA because gene expression can be detemined only by studying mRNA (but DNA represents a gene). We, therefore, isolate mRNA and use enzyme Reverse Transcriptase (mixed in the buffer for radiolabelling of DNA) which transcribes mRNA back to cDNA copy. This is then PCR amplified for a huge number of copies and placed on the DNA strands (single) on the microchip allowing them to hybridise (base-pairing with complementary strands). This we the hybridised strand emit the radioactivity through which we can detect the expression levels, expressing genes and also look for ways for gene therapy.

COST : Chip costs about 50$ to 2500$. The entire set –up would be around 100-200, 000$

Applications (gene therapy, e.g Menkes Disease) :

STEP 1 : This time use this technology to check if our mutant ATP7A producing truncated gene is due to exon skipping of the gene. STEP 2 : Use mRNA from both normal and diseased males; reverse transcribe them into cDNA; amplify cDNA using PCR; radio label them green (normal) and red (diseased) for easy detection. The colour Yellow would mean ‘normal and diseased share the same pattern there’. We should have prior knowledge of the standard exons. Following incubation of DNA on chip with cDNA (labelled), we observe something like this :

*We can then insert proper exons once we know the locations and sequences using computer tools and previous databases.

Small amounts of sample required

10) PRODUCTION OF Knock Out Mice (abnormal mice)

Title : A gene is knocked out in mice to study its effect and importance compared to normal progeny.

Introduction : A gene (in DNA) can either be on or off (naturally), but if a gene is artificially ‘switched off’ or inactivated or disrupted helps in studying genotype and phenotype.

Working : A gene in study is isolated (can be also from cDNA library), disrupted off (knocked-out gene) its normal function, cloned into a suitable vector that can be transferred to the mouse-stem cells (present in the blastocyst stage of mouse embryo) or directly to fertilised eggs, implanted in the mouse and allowed to form progeny. The changes that are observed in the new born progeny (50-50 chances, dominant gene and recessive gene) are carefully observed against the progeny where the particular gene is still functional. Through such methodology we get to know of a gene function and the changes that occur due to a change/error in the gene. The mouse containing the knocked-out gene product is known as a knock-out mouse and its progeny (knock-out gene dominant) too would have the same features (if the parent mouse survives).

COST : Producing such knockout mice costs 5, 000$ - 30, 000$ (Mouse, reagents, instruments, kits, vectors and breeding)

Applications (gene therapy, e.g. Menkes Disease) : To study importance of ATP7A gene

STEP 1 : Isolate the gene ATP7A from the cDNA library (actual gene, no introns). The gene is then disrupted so that it is rendered non-functional or mal-functional (can be done by inserting second gene, e.g antibiotic resistane that mouse usually lack, into the essential coding region). This construct can then be inserted into a Vector that is then injected into the Embryonic stem cells (Blastocyst). A new mouse progeny can be detected by certain phenotypic changes contrast to normal mouse. The affected mouse will have kinky hair, pigmentation, skeletal defects, etc relating to Menkes disease.

STEP 2 :

Mouse 1

Mouse Blastocys

Some have kinky hair, small structure

ADVANTAGES DISADVANTAGESStudy of gene functions Some features generally differ from humans, not

successful everytimeStudy of gene struture and regulating elements Producing such a mouse is expensive (also breeding)Study of mutations Maintenance of transgenic mice needs more skill

and staffDrug development for various diseases User has chances of contamination and thus

diseased