シャッフルDNAファミリーを経由して合成アデノ随伴ウイルス（AAV）遺伝子治療ベクターのエンジニアリングと進化

The overall goal of the following experiment is to create a highly diverse library of synthetic adeno associated viral capsids from which particles with a desired property can be selected. This is achieved by first fragmenting, cloned and selected a a v capsid genes via controlled DNAs one digestion to permit their subsequent reassembly into chimeric sequences via two PCR reactions. As a second step, the resulting PCR product is cloned back into a replication competent a a v construct to yield a plasmid library comprising at least 1 million different a a v capsid genes.

Next, this plasmid library is transfected into 2 9 3 T cells, together with an adenoviral helper plasmid in order to produce a chimeric virus library that can then be selected on target cells or in animals. The results show the successful molecular engineering and evolution of novel synthetic A A V vectors based on enhanced or more specific reporter gene expression in cultured cells or in adult mice. The main advantage of this technique over existing methods like peptide display is that DNA family shuffling has the potential to simultaneously alter and improve multiple features of the AV capsid from cell binding and intracellular trafficking to uncoating and gene expression in the nucleus.

This method can help answer key questions in the gene therapy and virology fields, such as how to obtain an ideal vector for a given therapeutic application or how to dissect the domains of a viral capsid that are responsible for a certain property. The implications of this technique extend towards therapy of multiple human diseases, including infection with viral pathogens because our method allows the molecular evolution of a A V vectors that specifically and potently infect viral target cells like T-cells for HIV or hepatocytes for hepatitis viruses. Though this method can provide insight into a V biology and can be used to improve AV vectors for gene therapy.

It can readily also be applied to other gene families of basic or applied relevance such as dose encoding fluorescence, three reporters, cellular enzymes or proteins involved in RNA interference. We first had the idea for this method when we were trying to find a good vector to transfuse slivers of adult mice with therapeutic genes and realize that all naturally occurring AV serotypes had disadvantages such as high prevalence in the human population and accordingly, high incidence of utilizing immunity or poor performance in vivo despite efficient transduction of culture. Hepatocytes in vitro Visual demonstration of this method is critical as the DNA based AV caps gene fragmentation and some of the viral library production steps are difficult to learn because they require experience and the awareness of the critical stages To facilitate routine preparation of sufficient amounts of the various adeno associated viral capsid genes.

For DNA shuffling these genes are initially sub cloned into a common plasmid backbone. To accomplish this, the desired cap genes are first amplified by PCR from commonly available A A V plasmids using primer's cap F and cap R.The cap genes are then cloned into a plasmid engineered to contain specific primer binding sites as well as restriction sites. In this example, the cap gene is cloned into the PAC one and a SC one sites, which are present in the primers used for cap amplification and in the recipient plasmid, but absent in most AAVs upon generation of the plasmid sets encoding A A V capsid genes begin the procedure for cap shuffling by PCR, amplifying the cap genes of choice using the T three and T seven primers.

Set up a 50 microliter PCR reaction containing 200 nanograms of cap plasmid, each primer at two micromolar final concentration, 10 microliters of five x high five buffer, and one microliter of high five polymerase. This reaction will yield about three micrograms of PCR product, which suffices for up to six shuffling reactions depending on the number of cap genes to be included in the library. Start with five minutes at 95 degrees Celsius and then run 40 cycles of 15 seconds at 94 degrees Celsius, 30 seconds at 57 degrees Celsius and three minutes at 68 degrees Celsius, followed by a final step of 10 minutes at 72 degrees Celsius.

After purifying the PCR products, A controlled DNA digest is used to create cap gene fragments for reassembly into Kyra. One of the most difficult aspects of this procedure is to obtain a certain useful range of DNA digested cap DNA fragments for the reassembly into kyras towards this aim, we prepare PLE amounts of purified cap genes to begin with and then set up multiple parallel reactions with varying conditions. To set up the digest equally, mix the various cap PCR products to a total amount of four micrograms in 54 microliters of water and then add six microliters DS reaction buffer and 0.5 microliters DNAs one to the reaction carefully the tube three times spin briefly and immediately put on a 25 degree Celsius heating block.

Incubate this first reaction for one minute. Next, prepare a second digest reaction, but incubate at 25 degrees Celsius for one minute and 15 seconds. In this way, set up multiple parallel reactions with incubation times between one and two minutes, varying the incubation time in increments of 15 seconds.

Stop each reaction by adding six microliters of 25 millimolar EDTA. Briefly vortex the tube and incubate for 10 minutes at 75 degrees Celsius. The multiple parallel reactions will result in five cap samples digested with DNAs, one for varying incubation times, purify the cap fragments on a standard 1%AROS gel.

Ideally, a smear should be visible between 100 and 500 base pairs as shown in this example with incubation times of one minute, 45 seconds or two minutes to begin. DNA family shuffling the cap fragments are first reassembled into full length sequences via A PCR in which they self prime based on partial homologies. Set up a 50 microliter reaction with 500 nanograms of purified fragments, 10 microliters of 10 x fusion buffer, one microliter of 10 millimolar dn tps, 1.5 microliters of DMSO and 0.5 microliters of fusion.

Two polymerase incubate for 30 seconds at 98 degrees Celsius and then run 40 cycles of 10 seconds at 98 degrees Celsius, 30 seconds at 42 degrees Celsius and 45 seconds at 72 degrees Celsius, followed by a final 10 minute step at 72 degrees Celsius. Next, amplify the reassembled cap genes for subsequent cloning by a second PCR using primers that bind to the conserved flanking sequences. For example, SAF and SAR or CUF and CUR set up a 50 microliter reaction containing two microliters of the first PCR each primer at a final concentration of two micromolar 0.5 microliters of magnesium chloride, 10 microliters of five x high-FI buffer and one microliter of high-FI polymerase.

16 to 24 PCRs are recommended to ensure sufficiently high yields for subsequent cloning. After pooling the PCRs and purifying the full length cap band, the next step is to digest the purified cap gene pool with PAC one and a SC one for cloning into a replication competent a a v plasmid. The recipient plasmid carries a a v inverted terminal repeats flanking the AAV two rep gene under the control of the AAV P five promoter, PAC one and ASC one.

Restriction sites downstream of rep allow for in-frame cloning of the pool of shuffled cap. The LSE primer binding sites indicated by arrows are useful for cap gene sequencing, ate the cap fragments and the appropriately cut recipient plasmid backbone at a three to one molar ratio. Make a 40 microliter total volume master mix sufficient for 20 transformations with a final DNA concentration of 15 nanograms per microliter.

Incubate overnight at 16 degrees Celsius on the following day, transform the ligation reaction into e coli. Mix two microliters ligation reaction with 30 microliters electro competent e coli and transfer into pre-cool electroporation cuvettes on ice electro at 1.8 kilovolts, 200 ohms and 25 microfarads. The time constant should be close to five milliseconds immediately at one milliliter Prewarm, SOC medium, and transferred to a 250 milliliter flask.

20 such electroporation will yield a library with a diversity of about one times 10 to the six different clones. Add one volume prewarm SOC medium to the pooled transformations and shake at 37 degrees Celsius and 180 RPM for one hour. Then add 40 milliliters of the pooled transformations to 800 milliliters of lb medium plus ampicillin and incubate for another 16 hours under the same conditions, the library plasma DNA is then purified using a commercially available kit.

Production of the viral library begins with the transfection of HEC 2 9 3 T cells with the a a v library to begin this procedure, seed 10 15 centimeter square dishes of HEC 2 9 3 T cells and grow them for 48 hours at 37 degrees Celsius. For each transfection, prepare the following two reagent mixtures in two separate tubes. The following quantities are given per 10 plates in the first tube.

Mix 7.9 milliliters, sodium chloride and DNA and add water to a total volume of 15.8 milliliters In the second tube, add 3.52 milliliters, polyethylene amine, 7.9 milliliters, sodium chloride, and 4.38 milliliters water. Combine the mixes vortex and incubate for 10 minutes at room temperature, distribute the solution evenly across each dish of he 2 9 3 T cells. Incubate at 37 degrees Celsius.

After 48 hours, the viral library can be harvested. Scrape off the cells into the medium, pool the cells from all the dishes in a single tube and spin down at 1200 RPM for 15 minutes. Resuspend the cell pellet in six milliliters, lysis, buffer, and then subject to five freeze thaw cycles after the five freeze thaw cycles, incubate with Ben Nase for one hour at 37 degrees Celsius.

Next, spin down cell debris at 3, 750 RPM for 20 minutes. Set up a gradient for a a v purification by using a paste pipette to add five milliliters virus suspension into a Beckman Quicks seal centrifuge tube, followed by 1.5 milliliters each of 15%25%and 40%idal solution. Top off the gradient with lysis buffer ultracentrifuge at 50, 000 K for two hours at four degrees Celsius.

At the completion of ultracentrifugation, remove the tube from the rotor and clean the outside of the tube with 70%ethanol. Stick a needle into the top of the tube for ventilation and use another needle to draw 1.2 milliliters of the 40%IOL fraction. Take care to avoid drawing from the 25%fraction as it contains empty.

A a v capsids. Individual clones from the original plasmid library are analyzed for their functionality and diversity by using them to produce recombinant. A a v vectors expressing a reporter gene cultured cells or animals are then infected with different amounts of the purified virus and transduction efficiency is determined.

Shown here are fluorescence microscopy results from three human cell lines infected with five different recombinant YFP expressing a a v vectors made with shuffled cap genes. An alternative method for analyzing transduction efficiency is facts-based measurement of fluorescent reporter gene expression. This figure shows a typical result from four cell types, human, kidney, human cervix, murine, fibroblasts, and human T cell infected with 18 different recombinant YFP expressing a V vectors made with shuffled cap genes randomly selected from an original library.

YFP expression was color coded for easier. Visualization depicted are percentages of transduced cells whereby black indicates 0%and white indicates the highest number measured in each cell type clone B two red exemplifies a clone with poor overall efficacy as can be expected from unselected capsids. The viral library can then be screened for single particles displaying most or all desired properties in a given cell line or in animals For selection.

In cultured cells, the cells are first co-infected with various aliquots of the library and ENO virus five to support a a v growth, then the cells are harvested and the amplified a a v is extracted and used to reinfect new cells. Determining optimal a a v adenovirus ratios for the co-infection of cells is important and a good measure for potent arius infection is the occurrence of cytopathic effects three days after virus inoculation evidenced by cell rounding and detaching in this figure, the top left panel shows uninfected cells while the other panels show cells infected with the indicated amounts in particles per cell of adenovirus. Another useful readout for a a v infection and amplification is detection of capsid proteins by western blotting using the B one antibody that recognizes a highly conserved a a v capsid epitope.

The left blot shows cells co-infected with various volumes in microliters of an a a V library and helper ENO vir. The first two lanes are good examples for conditions yielding barely detectable a a v protein expression indicating sufficient but not excessive, a a v infection and amplification, and hence the desired tight genotype phenotype linkage. Therefore, 0.11 or 10 microliters of these supernatants were used for reinfection of fresh cells.

Results from the western blot are shown on the right cells. In LA c were infected with a a v alone as a negative control. It is also critical to monitor library diversity during repeated infection rounds by DNA sequencing.

This diagram shows the comparisons of protein sequences of AAV clones from a library based on AAV two, eight and nine before and after selection sequences above the red line show the parental AAVs Purple arrows indicate homologous recombination events. The red arrow marks a crossover between AAV two and AAV nine noted in all selected clones. For selection in animals, the a a V library is infused into the animals rescued from the target cells or tissue by PCR and then re cloned and repackaged for a new infection round.

Shown here is an example of a rescue of successfully infected a a v clones from mouse livers one week after peripheral library infusion. Lane one shows the expected 2.2 kilobase band while lane two is a non template control sizes of DNA marker bands in lane M are indicated in kilobases. Regardless of the selection procedure, the enriched capsid variance must ultimately be validated in appropriate systems.

An example of superior performance of an enriched a a V kymera in cultured cells is shown here. Darker colors indicate higher infectivity is per particle number, hence clone. A-A-V-D-J outperforms a collection of eight natural a a v wild types in a broad range of cell lines.

Finally, an analysis of a selected a a V KYMERA in mouse liver is shown clone. A A V DN was selected on mirroring hepatoma cells and then used to produce lucid brace expressing recombinant vectors shown our representative mice three per group one week after peripheral infusion of equal doses of this vector or a control based on wild type AAV eight, one of the most potent known natural isolates in mouse liver. Note that while the A VDN clone gives slightly less overall expression in the liver, it is more specific for this organ since it exhibits substantially less off targeting in non hepatic tissues once the liver expression levels have been adjusted via the imaging software Once mastered.

This technique can be done in one week starting with the amplification of the a a V capsid genes and ending with the viral library if it is performed properly. While attempting this procedure, it's important to remember that a protocol provided here has been derived from our experiences with the selection of natural AV serotypes and that it is well possible that some of the conditions may need to be adopted and fine tuned when other users are making their own custom libraries. Besides this procedure, other methods like peptide display site directed with AGENESIS or PCR based randomization can be performed in order to also generate highly diverse libraries or to attempt to further improve the properties of capsids that have been created and enriched using DNA family shuffling After its development.

This technique paved the way for researchers in the field of viral gene delivery to explore therapeutic strategies for diseases and related target cells for which conventional A VC types provided unsatisfying results such as HIV infection and T cells, as well as to explore vector-based gene transfer into human stem cells and other heart to infect cells in culture. After watching this video, you should have a good understanding of how to engineer and evolve highly diverse libraries of synthetic adeno associated viral vectors using a combination of gene fragmentation and reassembly, as well as how to principally select these libraries in cultured cells or in animals in order to enrich individual capsids with desired specific properties for uses gene transfer vectors. Don't forget that working with adenovirus can be has and many precautions should be taken while performing this procedure.