Name: isolation of next-generation gene therapy vectors through engineering, barcoding, and screening of adeno-associated virus (aav) capsid variants
Uploaded: 2022-10-18T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants at JoVE.com

D.G. greatly appreciates support by the German Research Foundation (DFG) through the DFG Collaborative Research Centers SFB1129 (Projektnummer 240245660) and TRR179 (Projektnummer 272983813), as well as by the German Center for Infection Research (DZIF, BMBF; TTU-HIV 04.819).

1. AAV2 random 7-mer peptide display library preparation
NOTE: For the preparation of an AAV2 random peptide display library, synthesize the degenerate oligonucleotides as single-stranded DNA, convert it to double-stranded DNA, digest, ligate to the acceptor plasmid, and electroporate.
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	<li>Design of degenerate oligonucleotides
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		<li>Order the degenerate oligonucleotides and avoid codon bias. In the oligonucleotide 5&#39; CAGTCGGCCAG AG W GGC (X01)7 GCC...

<ol>
	<li>Wang, D., Tai, P. W. L., Gao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+vector+as+a+platform+for+gene+therapy+delivery.">Adeno-associated virus vector as a platform for gene therapy delivery.</a> Nature Reviews Drug Discovery. 18 (5), 358-378 (2019).</li><li>Muhuri, M., Levy, D. I., Schulz, M., McCarty, D., Gao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durability+of+transgene+expression+after+rAAV+gene+therapy.">Durability of transgene expression after rAAV gene therapy.</a> Molecular Therapy. 30 (4), 1364-1380 (2022).</li><li>Li, C., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+adeno-associated+virus+vectors+for+gene+therapy.">Engineering adeno-associated virus vectors for gene therapy.</a> Nature Reviews Genetics. 21 (4), 255-272 (2020).</li><li>Kuzmin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+landscape+for+AAV+gene+therapies.">The clinical landscape for AAV gene therapies.</a> Nature Reviews Drug Discovery. 20 (3), 173-174 (2021).</li><li>Mullard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+community+grapples+with+toxicity+issues,+as+pipeline+matures.">Gene therapy community grapples with toxicity issues, as pipeline matures.</a> Nature Reviews Drug Discovery. 20 (11), 804-805 (2021).</li><li>Nature Biotechnology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+at+the+crossroads.">Gene therapy at the crossroads.</a> Nature Biotechnology. 40 (5), 621 (2022).</li><li>Becker, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fantastic+AAV+Gene+Therapy+Vectors+and+How+to+Find+Them-Random+Diversification,+Rational+Design+and+Machine+Learning.">Fantastic AAV Gene Therapy Vectors and How to Find Them-Random Diversification, Rational Design and Machine Learning.</a> Pathogens. 11 (7), 756 (2022).</li><li>Perabo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+viral+vectors+with+modified+tropism:+the+adeno-associated+virus+display.">In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.</a> Molecular Therapy. 8 (1), 151-157 (2003).</li><li>Muller, O. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+peptide+libraries+displayed+on+adeno-associated+virus+to+select+for+targeted+gene+therapy+vectors.">Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.</a> Nature Biotechnology. 21 (9), 1040-1046 (2003).</li><li>Dalkara, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo-directed+evolution+of+a+new+adeno-associated+virus+for+therapeutic+outer+retinal+gene+delivery+from+the+vitreous.">In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous.</a> Science Translational Medicine. 5 (189), (2013).</li><li>Sahel, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+recovery+of+visual+function+in+a+blind+patient+after+optogenetic+therapy.">Partial recovery of visual function in a blind patient after optogenetic therapy.</a> Nature Medicine. 27 (7), 1223-1229 (2021).</li><li>Adachi, K., Enoki, T., Kawano, Y., Veraz, M., Nakai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drawing+a+high-resolution+functional+map+of+adeno-associated+virus+capsid+by+massively+parallel+sequencing.">Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.</a> Nature Communications. 5, 3075 (2014).</li><li>Marsic, D., Mendez-Gomez, H. R., Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-accuracy+biodistribution+analysis+of+adeno-associated+virus+variants+by+double+barcode+sequencing.">High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing.</a> Molecular Therapy-Methods &amp; Clinical Development. 2, 15041 (2015).</li><li>Korbelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+targeting+of+adeno-associated+viral+vectors+by+next-generation+sequencing-guided+screening+of+random+capsid+displayed+peptide+libraries.">Pulmonary targeting of adeno-associated viral vectors by next-generation sequencing-guided screening of random capsid displayed peptide libraries.</a> Molecular Therapy. 24 (6), 1050-1061 (2016).</li><li>Deverman, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-dependent+selection+yields+AAV+variants+for+widespread+gene+transfer+to+the+adult+brain.">Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.</a> Nature Biotechnology. 34 (2), 204-209 (2016).</li><li>Ravindra Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Cre-dependent+selection+yields+systemic+AAVs+for+targeting+distinct+brain+cell+types.">Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.</a> Nature Methods. 17 (5), 541-550 (2020).</li><li>Hanlon, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+an+efficient+AAV+vector+for+robust+CNS+transgene+expression.">Selection of an efficient AAV vector for robust CNS transgene expression.</a> Molecular Therapy-Methods &amp; Clinical Development. 15, 320-332 (2019).</li><li>Nonnenmacher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+blood-brain-barrier-penetrating+AAV+capsids+by+RNA-driven+biopanning.">Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning.</a> Molecular Therapy-Methods &amp; Clinical Development. 20, 366-378 (2021).</li><li>Tabebordbar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+a+family+of+AAV+capsid+variants+enabling+potent+muscle-directed+gene+delivery+across+species.">Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.</a> Cell. 184 (19), 4919-4938 (2021).</li><li>Davidsson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+capsid+evolution+approach+performed+in+vivo+for+the+design+of+AAV+vectors+with+tailored+properties+and+tropism.">A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.</a> Proceedings of the National Academy of Sciences. 116 (52), 27053-27062 (2019).</li><li>Pekrun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+barcoded+AAV+capsid+library+to+select+for+clinically+relevant+gene+therapy+vectors.">Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors.</a> Journal of Clinical Investigation Insight. 4 (22), (2019).</li><li>Ogden, P. J., Kelsic, E. D., Sinai, S., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+AAV+capsid+fitness+landscape+reveals+a+viral+gene+and+enables+machine-guided+design.">Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design.</a> Science. 366 (6469), 1139-1143 (2019).</li><li>Kondratov, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+study+of+a+29-capsid+AAV+library+in+a+non-human+primate+central+nervous+system.">A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system.</a> Molecular Therapy. 29 (9), 2806-2820 (2021).</li><li>Weinmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+myotropic+AAV+by+massively+parallel+in+vivo+evaluation+of+barcoded+capsid+variants.">Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.</a> Nature Communications. 11 (1), 5432 (2020).</li><li>Kremer, L. P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+screening+of+novel+AAV+capsids+identifies+variants+for+transduction+of+adult+NSCs+within+the+subventricular+zone.">High throughput screening of novel AAV capsids identifies variants for transduction of adult NSCs within the subventricular zone.</a> Molecular Therapy-Methods &amp; Clinical Development. 23, 33-50 (2021).</li><li>Borner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-arrayed+pan-AAV+peptide+display+libraries+for+rapid+single-round+screening.">Pre-arrayed pan-AAV peptide display libraries for rapid single-round screening.</a> Molecular Therapy. 28 (4), 1016-1032 (2020).</li><li>Kienle, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+evolution+of+synthetic+adeno-associated+virus+(AAV)+gene+therapy+vectors+via+DNA+family+shuffling.">Engineering and evolution of synthetic adeno-associated virus (AAV) gene therapy vectors via DNA family shuffling.</a> Journal of Visualized Experiments. (62), e3819 (2012).</li><li>Furuta-Hanawa, B., Yamaguchi, T., Uchida, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+droplet+digital+PCR+as+a+tool+for+titration+and+integrity+evaluation+of+recombinant+adeno-associated+viral+vectors.">Two-dimensional droplet digital PCR as a tool for titration and integrity evaluation of recombinant adeno-associated viral vectors.</a> Human Gene Therapy Methods. 30 (4), 127-136 (2019).</li><li>Lyons, E., Sheridan, P., Tremmel, G., Miyano, S., Sugano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+DNA+barcode+library+generation+for+biomolecule+identification+in+high-throughput+screens.">Large-scale DNA barcode library generation for biomolecule identification in high-throughput screens.</a> Scientific Reports. 7 (1), 13899 (2017).</li><li>Korbelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+design+and+production+strategies+for+novel+adeno-associated+viral+display+peptide+libraries.">Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.</a> Gene Therapy. 24 (8), 470-481 (2017).</li><li>Korbelin, J., Trepel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+successfully+screen+random+adeno-associated+virus+display+peptide+libraries+in+vivo.">How to successfully screen random adeno-associated virus display peptide libraries in vivo.</a> Human Gene Therapy Methods. 28 (3), 109-123 (2017).</li><li>Herrmann, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+and+all-inclusive+pipeline+for+shuffling+of+adeno-associated+viruses.">A robust and all-inclusive pipeline for shuffling of adeno-associated viruses.</a> American Chemical Society Synthetic Biology. 8 (1), 194-206 (2019).</li><li>Choudhury, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+selection+yields+AAV-B1+Capsid+for+central+nervous+system+and+muscle+gene+therapy.">In vivo selection yields AAV-B1 Capsid for central nervous system and muscle gene therapy.</a> Molecular Therapy. 24 (7), 1247-1257 (2016).</li><li>Buschmann, T., Bystrykh, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levenshtein+error-correcting+barcodes+for+multiplexed+DNA+sequencing.">Levenshtein error-correcting barcodes for multiplexed DNA sequencing.</a> BMC Bioinformatics. 14, 272 (2013).</li><li>Buschmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNABarcodes:+an+R+package+for+the+systematic+construction+of+DNA+sample+tags.">DNABarcodes: an R package for the systematic construction of DNA sample tags.</a> Bioinformatics. 33 (6), 920-922 (2017).</li><li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+mouse+transcriptomic+BodyMap+across+17+tissues+by+RNA-seq.">A comprehensive mouse transcriptomic BodyMap across 17 tissues by RNA-seq.</a> Scientific Reports. 7 (1), 4200 (2017).</li><li>Clarner, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+one-step+RT-ddPCR+method+to+determine+the+expression+and+potency+of+AAV+vectors.">Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors.</a> Molecular Therapy-Methods &amp; Clinical Development. 23, 68-77 (2021).</li><li>Zolotukhin, S., Vandenberghe, L. 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In this protocol, the steps needed for peptide display AAV capsid engineering and for barcoded AAV library screening, as well as for bioinformatic analysis of library composition and capsid performance, are outlined. This protocol focuses on the steps that facilitate the bioinformatic analysis of these types of libraries, because most virology laboratories lag in programming skills to match their proficiency in molecular biology techniques. Both types of libraries have been extensively described in the literature, as out...

Gene transfer therapy is the introduction of genetic material in cells to repair, replace, or alter the cellular genetic material to prevent, treat, cure, or ameliorate disease. Gene transfer, both in vivo and ex vivo, relies on different delivery systems, non-viral and viral. Viruses have evolved naturally to efficiently transduce their target cells and can be used as delivery vectors. Amongst the different types of viral vectors employed in gene therapy, adeno-associated viruses have been increasingly used, owing to their lack of pathogenicity, safety, low immunogenicity, and most importantly their ability to sustain long-term, non-integrating expression1,2,3. AAV gene therapy has yielded considerable achievements over the past decade; three therapies have been approved by the European Medicines Agency and the US Food and Drug Administration for use in humans3,4. Several clinical trials are also underway to treat a variety of diseases, such as hemophilia, muscular, cardiac, and neurological diseases, as reviewed elsewhere3. Despite decades of advancement, the field of gene therapy has experienced a series of setbacks in recent years4, most importantly deaths in clinical trials5 that have been put on hold due to dose-limiting toxicities, particularly for tissues that are massive, such as muscle, or difficult to reach, such as brain6.
The AAV vectors currently being used in clinical trials belong to the natural serotypes with a few exceptions1. AAV engineering offers the opportunity to develop vectors with superior organ- or cell-specificity and efficiency. In the past two decades, several approaches have been successfully applied, such as peptide display, loop-swap, capsid DNA shuffling, error-prone PCR, and targeted design, to generate individual AAV variants or libraries thereof&#160;with diverse properties7. These are then subjected to multiple rounds of directed evolution to select the variants within them with the desired properties, as reviewed elsewhere1,3. Of all the capsid evolution strategies, peptide display AAV libraries have been the most widely used, due to some unique properties: they are relatively easy to generate, and they can achieve high diversity and high-throughput sequencing, which allows trailing their evolution.
The first successful peptide insertion AAV libraries were described almost 20 years ago. In one of the first, Perabo et al.8 constructed a library of modified AAV2 capsids, in which a pool of randomly generated oligonucleotides was inserted in a plasmid at a position that corresponds to amino-acid 587 of the VP1 capsid protein, in the three-fold axis protruding from the capsid. Using adenovirus co-infection, the AAV library was evolved through multiple rounds of selection, and the final re-targeted variants were shown to be capable of transducing cell lines refractory to the parental AAV28. Shortly thereafter, M&#252;ller et al.9 introduced the two-step system for library production, a significant improvement to the protocol. Initially, the plasmid library, together with an adenoviral helper plasmid, are used to produce an AAV library that contains chimeric capsids. This AAV shuttle library is used to infect cells at low multiplicity of infection (MOI), with the aim to introduce one viral genome per cell. Co-infection with adenovirus ensures the production of AAVs with a matching genome and capsid9. About a decade later, Dalkara10 used in vivo directed evolution to create the 7m8 variant. This variant has a 10 amino-acid insertion (LALGETTRPA), three of which act as linkers, and efficiently targets the outer retina after intravitreal injection10. This engineered capsid is an exceptional success story, as it is one of the few engineered capsids to make it to the clinic thus far11.
The field experienced a second boost with the introduction of next-generation sequencing (NGS) techniques. Two publications from Adachi et al.12&#160;in 2014 and from Marsic et al.13&#160;in&#160;2015, showcased the power of NGS to track the distribution of barcoded AAV capsid libraries with high accuracy. A few years later, the NGS of barcoded regions was adapted to the peptide insertion region to follow the capsid evolution. K&#246;rbelin et al.14 performed an NGS-guided screening to identify a pulmonary-targeted AAV2-based capsid. The NGS analysis helped calculate three rating scores: the enrichment score between selection rounds, the general specificity score to determine tissue specificity, and finally the combined score14. The Gradinaru lab15 published the Cre-recombination-based AAV targeted evolution (CREATE) system in the same year, which facilitates a cell-type-specific selection. In this system, the capsid library carries a Cre-invertible switch, as the polyA signal is flanked by two loxP sites. The AAV library is then injected in Cre mice, where the polyA signal is inverted only in Cre+ cells, providing the template for binding of a reverse PCR primer with the forward primer within the capsid gene. This highly specific PCR rescue enabled the identification of the AAV-PHP.B variant that can cross the blood-brain barrier15. This system was further evolved into M-CREATE (Multiplexed-CREATE), in which NGS and synthetic library generation were integrated in the pipeline16.
An improved RNA-based&#160;version of this system from the Maguire lab17, iTransduce, allows selection on the DNA level of capsids that functionally transduce cells and express their genomes. The viral genome of the peptide display library comprises a Cre gene under the control of a ubiquitous promoter and the capsid gene under the control of the p41 promoter. The library is injected in mice that have a loxP-STOP-loxP cassette upstream of tdTomato. Cells transduced with AAV variants that express the viral genome and therefore Cre express tdTomato and, in combination with cell markers, can be sorted and selected17. Similarly, Nonnenmacher et al.18 and Tabebordbar et al.19 placed the capsid gene library under the control of tissue-specific promoters. After injection in different animal models, viral RNA was used to isolate the capsid variants.
An alternative approach is to use barcoding to tag capsid libraries. The Bj&#246;rklund lab20 used this approach to barcode peptide insertion capsid libraries and developed the barcoded rational AAV vector evolution (BRAVE). In one plasmid, the Rep2Cap cassette is cloned next to an inverted terminal repeats (ITR)-flanked, yellow fluorescent protein (YFP)-expressing, barcode-tagged transgene. Using loxP sites between the end of cap and the beginning of the barcode, an in vitro Cre recombination generates a fragment small enough for NGS, thereby allowing the association of peptide insertion with the unique barcode (look-up table, LUT). AAV production is performed using the plasmid library and the barcodes expressed in the mRNA are screened after in vivo application, again with NGS20. When the capsid libraries comprise variants of the whole capsid gene (i.e., shuffled libraries), long-read sequencing needs to be used. Several groups have used barcodes to tag these diverse libraries, which enables NGS with higher read depth. The Kay lab21 tagged highly diverse capsid shuffled libraries with barcodes downstream of the cap polyA signal. In a first step, a barcoded plasmid library was generated, and the shuffled capsid gene library was cloned into it. Then a combination of MiSeq (short read, higher read depth) and PacBio (long read, lower read depth) NGS as well as Sanger sequencing was used to generate their LUT21. In 2019, Ogden and colleagues from the Church lab22 delineated the AAV2 capsid fitness for multiple functions using libraries that had single point mutations, insertions, and deletions in every position, which ultimately enabled machine-guided design. For the generation of the library, smaller fragments of the capsid gene were synthesized, tagged with a barcode, next-generation sequenced, and then cloned into the full capsid gene. The NGS data were used to generate a LUT. The library was then screened using just the barcodes and short read sequencing, which in turn allows higher read depth22.
Barcoded libraries have been predominantly&#160;used to screen a pool of known, natural, and engineered variants following several rounds of selection of capsid libraries or independent of a capsid evolution study. The advantage of such libraries is the opportunity to screen multiple capsids, whilst reducing animal numbers and minimizing variation between animals. The first studies that introduced this technology to the AAV field were published almost a decade ago. The Nakai lab12 tagged 191 double alanine mutants covering amino acids 356 to 736 on the VP1 from AAV9 with a pair of 12-nucleotide barcodes. Using NGS, the library was screened in vivo for galactose binding and other properties12. Marsic and colleagues delineated the biodistribution of AAV variants using also a double-barcorded analysis 1 year later13. A more recent study in non-human primates compared the biodistribution in the central nervous system of 29 capsids using different routes of delivery23. Our lab has recently published barcoded AAV library screens of 183 variants that included natural and engineered AAVs. These screens on the DNA and RNA level led to the identification of a highly myotropic AAV variant24&#160;in mice as well as others displaying a high cell-type specificity in the mouse brain25.
Here, we describe the methodology used in this work and expand on it to include screening of AAV peptide display libraries. This comprises the generation of AAV2 peptide display libraries, a digital droplet PCR (dd-PCR) method for quantification, and finally an NGS pipeline to analyze the AAV variants, based in part on the work by Weinmann and colleagues24. Finally, a description of the generation of barcoded AAV libraries and the NGS pipeline used in the same publication is provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplification primer</td>
			<td>ELLA Biotech (Munich, Germany)</td>
			<td>-</td>
			<td>Second-strand synthesis of oligonucleotide insert</td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 Reagents</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>5067-1504</td>
			<td>DNA fragment validation</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>G2938C</td>
			<td>DNA fragment validation</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA Mini Kit&#160;</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>80204</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 Reagents</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>5067-1504</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>G2938C</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>BC-seq fw:&#160;</td>
			<td>IDT (San Joce, CA, CA, USA)</td>
			<td>ATCACTCTCGGCATGGACGAGC&#160;</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>BC-seq rv:&#160;&#160;</td>
			<td>IDT (San Joce, CA, CA, USA)</td>
			<td>GGCTGGCAACTAGAAGGCACA&#160;</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Millipore Sigma (Burlington, MA, USA)</td>
			<td>44-420-3250ML</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>BglI</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>R0143</td>
			<td>Digestion of double-stranded insert</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1851196</td>
			<td>dd-PCR cycler</td>
		</tr>
		<tr>
			<td>dNTPS&#160;</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>N0447S</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for probes (no dUTP)</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863024</td>
			<td>dd-PCR supermix</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863005</td>
			<td>dd-PCR droplet generation oil</td>
		</tr>
		<tr>
			<td>DG8 Cartridges for QX100 / QX200 Droplet Generator</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1864008</td>
			<td>dd-PCR droplet generation cartridge</td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863051</td>
			<td>dd-PCR cartridge holder</td>
		</tr>
		<tr>
			<td>Droplet Generator DG8 Gasket</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863009</td>
			<td>dd-PCR cover for cartridge</td>
		</tr>
		<tr>
			<td>ddPCR Plates 96-Well, Semi-Skirted</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>12001925</td>
			<td>dd-PCR 96-well plate</td>
		</tr>
		<tr>
			<td>E.cloni 10G SUPREME Electrocompetent Cells</td>
			<td>Lucigen (Middleton, WI, USA)</td>
			<td>60081-1</td>
			<td>Electrocompetent cells</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes, 1mm</td>
			<td>Biozym Scientific (Oldendorf, Germany)</td>
			<td>748050</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>GAPDH primer/probe mix</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>Mm00186825_cn</td>
			<td>Taqman qPCR primer</td>
		</tr>
		<tr>
			<td>Genepulser Xcell</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1652660</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems (Waltham, MA, USA)</td>
			<td>4368814</td>
			<td>cDNA reverse transcription</td>
		</tr>
		<tr>
			<td>ITR_fw</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>GGAACCCCTAGTGATGGAGTT (https://signagen.com/blog/2019/10/25/qpcr-primer-and-probe-sequences-for-raav-titration/)</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>ITR_rv</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>CGGCCTCAGTGAGCGA (https://signagen.com/blog/2019/10/25/qpcr-primer-and-probe-sequences-for-raav-titration/)</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>ITR_probe</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>HEX-CACTCCCTCTCTGCGCGCTCG-BHQ1 (https://signagen.com/blog/2019/10/25/qpcr-primer-and-probe-sequences-for-raav-titration/)</td>
			<td>dd-PCR probe</td>
		</tr>
		<tr>
			<td>Illumina NextSeq 500 system</td>
			<td>Illumina Inc (San Diego, CA, USA)</td>
			<td>SY-415-1001</td>
			<td>NGS Library sequencing</td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix (2X)*</td>
			<td>Roche AG (Basel, Switzerland)</td>
			<td>KK2600 07958919001</td>
			<td>NGS sample prepration</td>
		</tr>
		<tr>
			<td>MagnaBot 96 Magnetic Separation Device</td>
			<td>Promega GmbH (Madison, WI, USA)</td>
			<td>V8151</td>
			<td>Sample prepration for NGS library</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 spectrophotometer</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>ND-2000</td>
			<td>Digestion of double-stranded insert</td>
		</tr>
		<tr>
			<td>NGS_frw</td>
			<td>Sigma-Aldrich (Burlinght, MA, USA)</td>
			<td>GTT CTG TAT CTA CCA ACC TC</td>
			<td>NGS primer</td>
		</tr>
		<tr>
			<td>NGS_rev</td>
			<td>Sigma-Aldrich (Burlinght, MA, USA)</td>
			<td>CGC CTT GTG TGT TGA CAT C</td>
			<td>NGS primer</td>
		</tr>
		<tr>
			<td>NextSeq 500/550 High Output Kit (75 cycles)</td>
			<td>Illumina Inc (San Diego, CA, USA)</td>
			<td>FC-404-2005</td>
			<td>NGS Library sequencing</td>
		</tr>
		<tr>
			<td>Ovation Library System for Low Complexity Samples Kit&#160;</td>
			<td>NuGEN Technologies, Inc. (San Carlos, CA, USA)</td>
			<td>9092-256</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>PX1 Plate Sealer&#160;</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1814000</td>
			<td>dd-PCR plate sealer</td>
		</tr>
		<tr>
			<td>Pierceable Foil Heat Seal</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1814040</td>
			<td>dd-PCR sealing foil</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA-Polymerase</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>F530S</td>
			<td>Second-strand synthesis of oligonucleotide insert</td>
		</tr>
		<tr>
			<td>PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)</td>
			<td>Polysciences, Inc. (Warrington, PA, USA)</td>
			<td>24765-1G</td>
			<td>AAV library preparation</td>
		</tr>
		<tr>
			<td>ProNex Size-Selective Purification System</td>
			<td>Promega GmbH (Madison, WI, USA)</td>
			<td>NG2002</td>
			<td>Sample prepration for NGS library</td>
		</tr>
		<tr>
			<td>Phusion Hot Start II Polymerase&#160;</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>F549L</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche AG (Basel, Switzerland)</td>
			<td>5963117103</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>pRep2Cap2_PIS</td>
			<td></td>
			<td></td>
			<td>ITR-Rep2Cap2-ITR vector. Peptide insertion site within the Cap2 ORF, manufactured/prepared in the lab&#160;</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1864002</td>
			<td>dd-PCR droplet generator</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1864003</td>
			<td>dd-PCR droplet analysis</td>
		</tr>
		<tr>
			<td>QIAquick Nucleotide Removal Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>28306</td>
			<td>Second-strand synthesis of oligonucleotide insert purification</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>28704</td>
			<td>Plasmid vector purification</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>12162</td>
			<td>Plasmid library DNA preparation</td>
		</tr>
		<tr>
			<td>Qiaquick PCR Purification kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>28104</td>
			<td>Sample prepration for NGS library</td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>Invitrogen (Waltham, MA, USA)</td>
			<td>Q32857</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>Q32851</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>QuantiFast PCR Master Mix</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>1044234</td>
			<td>Taqman qPCR</td>
		</tr>
		<tr>
			<td>rep_fw</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>AAGTCCTCGGCCCAGATAGAC</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>rep_rv</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>CAATCACGGCGCACATGT</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>rep_probe</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>FAM-TGATCGTCACCTCCAACA-BHQ1</td>
			<td>dd-PCR probe</td>
		</tr>
		<tr>
			<td>RNase-free DNase</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>79254</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>SfiI</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>R0123</td>
			<td>Digestion of vector</td>
		</tr>
		<tr>
			<td>5 mm, steel Beads</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>69989</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>TRIMER-oligonucleotides</td>
			<td>ELLA Biotech (Munich, Germany)</td>
			<td>-</td>
			<td>Degenerate oligonucleotide</td>
		</tr>
		<tr>
			<td>T4 Ligase</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>M0202L</td>
			<td>Plasmid library ligation</td>
		</tr>
		<tr>
			<td>TissueLyserLT</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>85600</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>YFP_fw</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>GAGCGCACCATCTTCTTCAAG</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>YFP_rv</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>TGTCGCCCTCGAACTTCAC</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>YFP_probe</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>FAM-ACGACGGCAACTACA-BHQ1</td>
			<td>dd-PCR probe</td>
		</tr>
		<tr>
			<td>Zymo DNA Clean &#38; Concentrator-5 (Capped)</td>
			<td>Zymo research (Irvine, CA, USA)</td>
			<td>D4013</td>
			<td>Vector and Ligation purification</td>
		</tr>
	</tbody>
</table>

isolation of next-generation gene therapy vectors through engineering, barcoding, and screening of adeno-associated virus (aav) capsid variants

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by encouraging clinical data and the approval of several AAV gene therapies. Two major reasons for the success of AAV vectors are (i) the prior isolation of various naturally occurring viral serotypes with distinct properties, and (ii) the subsequent establishment of powerful technologies for their molecular engineering and repurposing in high throughput. Further boosting the potential of these techniques are recently implemented strategies for barcoding selected AAV capsids on the DNA and RNA level, permitting their comprehensive and parallel in vivo stratification in all major organs and cell types in a single animal. Here, we present a basic pipeline encompassing this set of complementary avenues, using AAV peptide display to represent the diverse arsenal of available capsid engineering technologies. Accordingly, we first describe the pivotal steps for the generation of an AAV peptide display library for the in vivo selection of candidates with desired properties, followed by a demonstration of how to barcode the most interesting capsid variants for secondary in vivo screening. Next, we exemplify the methodology for the creation of libraries for next-generation sequencing (NGS), including barcode amplification and adaptor ligation, before concluding with an overview of the most critical steps during NGS data analysis. As the protocols reported here are versatile and adaptable, researchers can easily harness them to enrich the optimal AAV capsid variants in their favorite disease model and for gene therapy applications.

Generation of an AAV2 peptide display library. As a first step toward the selection of engineered AAVs, the generation of a plasmid library is described. The peptide insert is produced by using degenerate primers. Reducing the combination of codons in those from 64 to 20 has the advantages of eliminating stop codons and facilitating NGS analysis, by reducing library diversity on the DNA but not the protein level. The oligonucleotide insert is purchased as single-stranded DNA (Figure ...

Watch this Scientific Journal Video about Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants at JoVE.com

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants

AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs.

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by ...

This protocol closes a significant gap between natural evolution of viruses and their use as recombinant vectors for human gene therapy by enabling their engineering, diversification, and stratification. This technique allows for the high-throughput parallel screening for novel, isolated, or engineered adeno-associated virus, in short AAV, variants, thereby saving on animal numbers, work, cost, and time. The identified AVV capsid variants can increase efficacy and specificity of AAV-based delivery of therapeutic transgenes, thereby reducing the required viral dose.This could improve safety and applicability of gene therapy. The same approach could also be used for capsid or promoter engineering of other gene delivery vehicles which improves our understanding of their biology and their use in gene therapy. Helping to demonstrate the procedure will be PhD students Jonas Becker and Jixin Liu, post-doc Joanna Szumska and Margarita Zayas, as well as research assistants Emma Gerstmann and Ellen Widtke from my laboratory.Begin by analyzing the NGS sequencing data with Python 3 and Biopython. The NGS analysis is composed of two steps. For the first step, search the sequence files for sequences that satisfy specific criteria such as flanking sequences, length, and location using script one, and a configuration file that provides the information needed.For the second step, translate the extracted sequences starting at the AGWGGC sequence using script number two and the configuration and zuordnung. txt files that provides the information needed. Prepare two folders:script and data.Copy the Gzip compressed files resulting from the sequencing to the data folder. Then copy the Python and configuration files to the script folder as described in the text manuscript. Before running the scripts, open the zuordnung.txt file and add two tab separated columns. Enter the names of the Gzip files in column one and the desired final name in column two. Change the variables in the configuration file as described in the text manuscript.Using the specific command, initiate the variant sequence detection and extraction. The output will be TXT files with the extracted DNA sequences and their numbers of reads. The header of this file contains statistical data and these data will be transferred to the following files.These TXT data will be the input file for script number two in which the DNA sequences are translated, ranked, and analyzed. Using the specific command, initiate PV translation and analysis of the text output files of script one as described in the text manuscript. The output files of script number two will be named using the second column of the lookup table in zuordnung.txt with extensions based on the type of analysis. Ensure that the three output files contain statistical data in the first rows and a first column with the index of each DNA sequence from the input text files. The remaining columns should consist of the DNA sequence, number of reads, forward or reverse read, and translated peptide sequence.The invalid sequences should have NA and not valid in the last two columns. Visualize the output files using available software based on the user's needs. To perform the analysis of the NGS sequencing data using custom code in Python 3, the workflow comprises the detection of barcode sequences guided by flanking sequences, their length and location, as well as analysis of barcode enrichment and distribution over the set of tissues.Prepare two folders:script and data. Copy the Gzip compressed files resulting from the sequencing to the data folder. Then copy the python and configuration files to the script folder as described in the text manuscript.Before executing the script, create two tab delimited text files:the capsidvariance. txt file with the barcode sequences assigned to AAV capsid variant names, and the contamination. txt file with barcode sequences that come from possible contamination.Finally, edit the configuration file to include the information of the path to folder with sequencing data, sequence of flanking regions of the barcodes, their position, and window size for barcode detection. Execute the barcode detection script with the provided paths and configuration files using the respective command. The output of this command execution will be TXT files with recounts per capsid variant and the total number of reads recovered from the raw data.To evaluate the distribution of barcoded AAV capsid among tissues or organs, in the zuordnung. txt file, assign the name of each text file obtained from the barcode detection run to a tissue or organ name. Add the names of text files in the first column and corresponding tissue or organ names in the tab delimited assignment.Create an organs. txt file with the list of names for on and off-target organs, which correspond to the names given in the assignment zuordnung. txt file.Then create normalization_organ. txt and normalization_variant. txt tab delimited text files with normalized values for all capsid variants and all organs and tissues.In the first column with the normalization_organ. txt file, write the names given for each organ, and the second column with normalization values for the corresponding tissue. Fill the first column of the normalization_variant.txt file with the list of capsid names, and the second column with the normalized values of the read counts for each capsid in the pooled library. Edit the configuration file by specifying the full paths to all additional files and execute the barcode analysis script using this specific command. The barcode analysis script outputs several files such as the text files with relative concentration or RC values of capsid distribution within different tissues based on multiple normalization steps described earlier, and the spreadsheet file which combines text file data into merged matrix data.Visualize the data and perform cluster analysis of the matrix data as described in the text manuscript. The script will input the relativeconcentration. xls files and generate two plots of hierarchical cluster heat map and principal component analysis.To modify plots or PNG parameters, open the R script and follow the instructions in the comment section. The quantified regions of the AAV2 rep gene showed that 99.2%were positive for ITR, suggesting that the AAV capsids contained the entire viral genomes. In all three sets, the extracted reads based on signature sequences specific to the library represented about 94%of the total reads, indicating good quality.Of these, more than 99%were valid PV reads and over 99%of the valid PV reads were unique, suggesting that the library was balanced with high internal diversity. The two major branches of the heat map hierarchy reflected the difference in the transduction efficiency of capsid variants. The left branch with the majority of the capsid variants included all the capsids which showed a high relative concentration value across most tissues.Apart from strikingly high liver specificity, three other capsids exhibited specificity in the diaphragm, skeletal muscle, biceps, and brain. The right branch of the hierarchical clustering included the capsid variants with overall lower transduction efficiency most evident in the duodenum and pancreas. The principal component analysis for the original subset forms the cluster of capsid variants with high liver specificity and outlines the VAR60 capsid outstanding muscle tropism.When attempting this protocol, you need to pay attention to typing errors. The syntax is also different if you're using the Windows command prompt compared to Linux. Following the identification of novel AAV variants, the therapeutic and translational potential needs to be verified in a deceased or larger animal model.This technique enables a direct side-by-side comparison of AAV variants under identical conditions and is extremely versatile, paving the way for its translation in large animals, or even in humans.

isolation-next-generation-gene-therapy-vectors-through-engineering

Research

JoVE Journal

Bioengineering

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while &#34;Candidatus Liberibacter asiaticus&#34; has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California&#39;s citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step.
Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/&#181;L) that was comparable with the CCPP&#39;s hand-chopping protocol (77.84 ng/&#181;L). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

We engineered, fabricated, and validated an instrument that rapidly processes phloem-rich bark citrus budwood tissues. Compared to current methods, the budwood tissue extractor (BTE) has increased sample throughput and decreased the required labor and equipment costs.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...