AAV Vector Engineering

Available here

Details here

M-CREATE - for isolating novel AAV variants from large viral libraries

Ravindra Kumar, et al., Nature Methods 2020 [Github ]

AAV-PHP Production - for individual virus preparations

Challis et al., Nature Protocols 2019 [ Protocols.io link ]

AAV Production & Administration Worksheets - for individual virus preparations

Transfection Calculator

Titration Calculator

Viral Injection Volume Calculator

• The CLOVER Center is equipped with a BSL-2 virus production facility as well as wet labs for molecular cloning, tissue processing, and macromolecular staining procedures.

• CLOVER offers collaborative custom vector development.

• One-on-one consultations and training for viral vector production to jump start research projects

• Focused workshops for researchers with various backgrounds and experience levels

• Yearly 5-day course on systemic AAVs and CLARITY as part of the NeuroNex MINT Technology Hub

Can be found here

• Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain. Chuapoco M.R., Flytzanis N.C., Goeden N., Octeau J.C., Roxas K.M., Chan K.Y., Scherrer J., Winchester J., Blackburn R.J., Campos L.J., Mimi Man K.N., Sun J., Chen X., Lefevre A., Singh V.P., Arokiaraj C.M., Shay T.F., Vendemiatti J., Jang M.J., Mich J.K., Bishaw Y., Gore B.B., Omstead V., Taskin N., Weed N., Levi B.P., Ting J.T., Miller C.T., Deverman B.E., Pickel J., Tian L., Fox A.S., Gradinaru V. Nature Nanotechnology, 2023; 10.1038/s41565-023-01419-x. [ Article ]

• Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates. Chen X., Wolfe D.A., Sivadasan Bindu D., Zhang M., Taskin N., Goertsen D., Shay T.F., Sullivan E.E., Huang S., Ravindra Kumar S., Arokiaraj C.M., Plattner V.M., Campos L.J., Mich J.K., Monet D., Ngo V., Ding X., Omstead V., Weed N., Bishaw Y., Gore B.B., Lein E.S., Akrami A., Miller C., Levi B.P., Keller A., Ting J.T., Fox A.S., Eroglu C., Gradinaru V. Nature Communications, 2023; 10.1038/s41467-023-38582-7. [ Article ]

• Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors. Shay T.F., Sullivan E.E., Ding X., Chen X., Ravindra Kumar S., Goertsen D., Brown D., Crosby A., Vielmetter J., Borsos M., Wolfe D.A., Lam A.W., Gradinaru V. Science Advances, 2023; 10.1126/sciadv.adg6618. [ Article | Caltech News Release ]

• Spatial transcriptomics for profiling the tropism of viral vectors in tissues. Jang M.J., Coughlin G.M., Jackson C.R., Chen X., Chuapoco M.R., Vendemiatti J.L., Wang A.Z., Gradinaru V. Nature Biotechnology, 2023; 10.1038/s41587-022-01648-w. [ Article | Research Briefing ]

• Molecular basis of astrocyte diversity and morphology across the CNS in health and disease. Endo F., Kasai A., Soto J.S., Qu Z., Hashimoto H., Gradinaru V., Kawaguchi R., Khakh B.S. Science, 2022; https://doi.org/10.1126/science.adc9020. [ Article | Perspective ]

• Multimodal imaging of capsid and cargo reveals differential brain targeting and liver detargeting of systemically-administered AAVs. Seo J.W., Ajenjo J., Wu B., Robinson E., Nura Raie M., Wang J., Tumbale S.K., Buccino P., Alexander Anders D., Shen B., Habte F.G., Beinat C., James M.L., Taylor Reyes S., Ravindra Kumar S., Miles T.F., Lee J.T., Gradinaru V., Ferrara K.W. Biomaterials, 2022; https://doi.org/10.1016/j.biomaterials.2022.121701. [ Article ]

• Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid. Goertsen D., Goeden N., Flytzanis N.C., Gradinaru V. Molecular Therapy Methods Clinical Development, 2022; https://doi.org/10.1016/j.omtm.2022.07.010. [ Article ]

• Structural basis of receptor usage by the engineered capsid AAV-PHP.eB. Jang S., Shen H.K., Ding X., Miles T.F., Gradinaru V. Molecular Therapy Methods Clinical Development, 2022; https://doi.org/10.1016/j.omtm.2022.07.011. [ Article ]

• Transduction of systemically administered adeno-associated virus in the colonic enteric nervous system and c-Kit cells of adult mice. Wang L., Yuan P., Challis C., Ravindra Kumar S., Tache Y. Frontiers in Neuroanatomy, 2022; https://doi.org/10.3389/fnana.2022.884280. [ Article ]

• Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems. Chen X., Ravindra Kumar S., Adams C.A., Yang D., Wang T., Wolfe D.A., Arokiaraj C.M., Ngo V., Campos L.J., Griffiths J.A., Ichiki T., Mazmanian S.K., Osborne P.B., Keast J.R., Miller C.T., Fox A.S., Chiu I.M., Gradinaru V. Neuron, 2022; https://doi.org/10.1016/j.neuron.2022.05.003. [ Article ]

• Glutamate in primary afferents is required for itch transmission. Cui L., Guo J., Cranfill S.L., Gautam M., Bhattarai J., Olson W., Beattie K., Challis R.C., Wu Q., Song X., Raabe T., Gradinaru V., Ma M., Liu Q., Luo W. Neuron, 2022; doi: 10.1016/j.neuron.2021.12.007. [ Article ]

• AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Goertsen D., Flytzanis N.C., Goeden N., Chuapoco M.R., Cummins A., Chen Y., Fan Y., Zhang Q., Sharma J., Duan Y., Wang L., Feng G., Chen Y., Ip N.Y., Pickel J., Gradinaru V. Nature Neuroscience, 2022; doi: 10.1038/s41593-021-00969-4. [ Article ]

• Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing. Brown D., Altermatt M., Dobreva T., Chen S., Wang A., Thomson M., Gradinaru V. Frontiers in Immunology, 2021; doi: 0.3389/fimmu.2021.730825. [ Article ]

• Improved systemic AAV gene therapy with a neurotrophic capsid in Neimann-Pick disease type C1 mice. Davidson C.D., Gibson A.L., Gu T., Baxter L.L., Deverman B.E., Beadle K., Incao A.A., Rodriguez-Gil J.L., Fujiwara H., Jiang X., Chandler R.J., Ory D.S., Gradinaru V., Venditti C.P., Paven W.J. Life Science Alliance, 2021; doi: 10.26508/lsa.202101040. [ Article ]

• Brain-wide Cas9-mediated cleavage of a gene causing familial Alzheimer's disease alleviates amyloid-related pathologies in mice. Duan Y., Ye T., Qu Z., Chen Y., Miranda A., Zhou X., Lok K.C., Chen Y., Fu A.K.Y., Gradinaru V., Ip N.Y. Nature Biomedical Engineering, 2022; doi: 10.1038/s41551-021-00759-0. [ Article ]

• Specific and behaviorally conseqeuntial astrocyte Gq GPCR signaling attenuation in vivo with ißark. Nagai J., Bellafard A., Qu Z., Yu X., Ollivier M., Gangwani M.R., Diaz-Castro B., Coppola G., Schumacher S.M., Golshani P., Gradinaru V., Khakh B.S. Neuron, 2021; doi: 10.1016/j.neuron.2021.05.023. [ Article ]

• Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery. Smith, E.J., Farshim, P.P., Flomen, R., Jones S.T., McAteer, S.J., Deverman B.E., Gradinaru V., Bates G.P. Brain Communications, 2021; doi: 10.1093/braincomms/fcab105. [ Article ]

• Multicolor sparse viral labeling and 3D digital tracing of the enteric plexus in mouse proximal colon using a novel adeno-associated virus capsid. Wang, L., Challis, C., Li, S., Fowlkes, C.C., Ravindra Kumar, S., Yuan, P.Q., Tache, Y.F. Neurogastroenterology & Motility, 2020; doi: 10.1111/nmo.14014. [ Article ]

• Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation. Seo, J. W., Ingham, E. S., Mahakian, L., Tumbale, S., Wu, B., Aghevlian, S., Shams, S., Baikoghli, M., Jain, P., Ding, X., Goeden, N., Dobreva, T., Flytzanis, N. C., Chavez, M., Singhal, K., Leib, R., James, M. L., Segal, D. J., Cheng, R. H., Silva, E. A., Gradinaru, V., Ferrara, K. W. Nature Communications, 2020; doi: 10.1038/s41467-020-15818-4.  [ Article ]

• Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Ravindra Kumar, S., Miles, T. F., Chen, X., Brown, D., Dobreva, T., Huang, Q., Ding, X., Luo, Y., Einarsson, P. H., Greenbaum, A., Jang, M. J., Deverman, B. E., Gradinaru, V. Nature Methods, 2020; doi: 10.1038/s41592-020-0799-7.  [ Article | Cover | Caltech News Release ]

• LRP1 is a master regulator of tau uptake and spread. Rauch, J. N., Luna, G., Guzman, E., Audouard, M., Challis, C., Sibih, Y., Leshuk, C., Hernandez, I., Wegmann, S., Hyman, B. T., Gradinaru, V., Kampmann, M., Kosik, K. S. Nature, 2020; doi: 10.1038/s41586-020-2156-5.  [ Article ]

• Gut-seeded α-Synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice. Challis, C., Hori, A., Sampson, T. R., Yoo, B. B., Challis, R. C., Hamilton, A. M., Mazmanian, S. K., Volpicelli-Daley, L. A., Gradinaru, V. Nature Neuroscience, 2020; doi: 10.1038/s41593-020-0589-7.  [ Article | Open access link | Caltech News Release ]

• Machine learning-guided channelrhodopsin engineering enables minimally invasive optogenetics. Bedbrook, C. N., Yang, K. K., Robinson, J. E., Mackey, E. D., Gradinaru, V., & Arnold, F. H. Nature Methods, 2019; doi: 10.1038/s41592-019-0583-8.  [ Article | Open access link | Caltech News Release ]

• Optical dopamine monitoring with dLight1 reveals mesolimbic phenotypes in a mouse model of neurofibromatosis type 1. Robinson, J. E., Coughlin, G. M., Hori, A. M., Cho, J. R., Mackey, E. D., Turan, Z., Patriarchi, T., Tian, L., Gradinaru, V. eLife, 2019; doi: 10.7554/eLife.48983.  [ Article ]

• Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies. Rajendran, P. S., Challis, R. C., Fowlkes, C. C., Hanna, P., Tompkins, J. D., Jordan, M. C., Hiyari, S., Gabris-Weber, B. A., Greenbaum, A., Chan, K. Y., Deverman, B. E., Münzberg, H., Ardell, J. L., Salama, G., Gradinaru, V., Shivkumar, K. Nature Communications, 2019; doi: 10.1038/s41467-019-09770-1.  [ Article ]

• TRIM9-Mediated Resolution of Neuroinflammation Confers Neuroprotection upon Ischemic Stroke in Mice. Zeng, J., Wang, Y., Luo, Z., Chang, L.-C., Yoo, J. S., Yan, H., Choi, Y., Xie, X., Deverman, B. E., Gradinaru, V., Gupton, S. L., Zlokovic, B. V., Zhao, Z., Jung, J. U. Cell Reports, 2019; doi: 10.1016/j.celrep.2018.12.055.  [ Article ]

• Epigenetic landscape and AAV targeting of human neocortical cell classes. Mich, J., Hess, E., Graybuck, L., Somasundaram, S., Miller, J., Ding, Y., Shapovalova, N., Fong, O., Yao, S., Mortrud, M., Chong, P., Bertagnolli, D., Goldy, J., Casper, T., Kroll, M., Hodge, R., Bakken, T., Yao, Z., Dee, N., Cetin, A., Smith, K., Gwinn, R., Cobbs, C., Ko, A., Ojemann, J., Keene, C., Silbergeld, D., Gradinaru, V., Sunkin, S., Zeng, H., Lein, E., Tasic, B., Ting, J. and Levi, B. bioRxiv, 2019; doi: 10.1101/555318.  [ Article ]

• Systemic AAV vectors for widespread and targeted gene delivery in rodents. Challis, R. C., Kumar, S. R., Chan, K. Y., Challis, C., Beadle, K., Jang, M. J., Kim, H. M., Rajendran, P. S., Tompkins, J. D., Shivkumar, K., Deverman, B. E., Gradinaru, V. Nature Protocols, 2019; doi: 10.1038/s41596-018-0097-3. [ Article | Cover | Open access link | High resolution fluorescent images ]

• The Neuropeptide Tac2 Controls a Distributed Brain State Induced by Chronic Social Isolation Stress Moriel Zelikowsky, May Hui, Tomomi Karigo, Andrea Choe, Bin Yang, Mario R. Blanco, Keith Beadle, Viviana Gradinaru, Benjamin E. Deverman and David J. Anderson. Cell 2018 May 17; doi: 10.1016/j.cell.2018.03.037.  [ Article | Open access link | Caltech News Release ]

• Viral Strategies for Targeting the Central and Peripheral Nervous Systems Bedbrook CN, Deverman BE, Gradinaru V. . Annu Rev Neurosci. 2018 Apr 25. doi: 10.1146/annurev-neuro-080317-062048.  [ Article ]

• Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Chan, K.Y., Jang, M.J., Yoo, B.B., Greenbaum, A., Ravi, N., Wu., W.L., Sanchez-Guardado, L., Lois, C., Mazmanian, S.K., Deverman, B.E., Gradinaru, V. Nature Neuroscience, 2017; doi:10.1038/nn.4593.  [ Article | Caltech News Release | News and Views | Cover | Open access link ]

• Global representations of goal-directed behavior in distinct cell types of mouse neocortex. Allen, W.E., Kauvar, I.V., Chen, M.Z., Richman, E.B., Yang, S.J., Chan, K., Gradinaru, V., Deverman, B.E., Luo, L., Deisseroth, K. Neuron, 2017;94, 891-907.  [ Article | DARPA Highlight ]

• Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Deverman, B.E.*; Pravdo, P.L.; Simpson, B.P.; Kumar, S.R.; Chan, K.Y.; Banerjee, A.; Wu, W.L.; Yang, B.; Huber, N.; Pasca, S.P.; Gradinaru, V.*, Nat Biotech, 2016; doi:10.1038/nbt.3440.  [ Article | Caltech News Release | Video | CIRM Blog | F1000 Exceptional]

56 Publications by other groups using our systemic AAV viruses in FY2022 – 5 highlighted

• Constitutively active SARM1 variants that induceneuropathy are enriched in ALS patients. A.J. Bloom, X. Mao, A. Strickland, Y.Sasaki, J. Milbrandt, A. DiAntonio Molecular Neurodegeneration 2022; 10.1186/s13024-021-00511-x.

• Regulated control of gene therapies by drug-induced splicing. A.M. Monteys, A.A. Hundley, P.T. Ranum, L. Tecedor, A. Muehlmatt, E. Lim, D. Lukashev, R. Sivasankaran, B.L. Davidson Nature 2021; 10.1038/s41586-021-03770-2.

• Functional diversity for body actions in the mesencephalic locomotor region. M.J.Ferreira-Pinto, H. Kanodia, A. Falasconi, M. Sigrist, M.S. Esposito, S. Arber Cell 2021; 10.1016/j.cell.2021.07.002.

• Severe deficiency of the voltage-gated sodium channel Nav1.2 elevates neuronal excitability in adult mice. J. Zhang, X. Chen, M.Eaton, J. Wu, Z. Ma, S. Lai, A. Park, T.S. Ahmad, Z. Que, J.H. Lee, T. Xiao, Y.Li, Y. Wang, M.I. Olivero-Acosta, J.A. Schaber, K. Jayant, C. Yuan, Z. Huang,N. Lanman, W.C. Skarnes, Y. Yang Cell Reports 2021; 10.1016/j.celrep.2021.109495.

• Noradrenergic terminal short-term potentiation enables modality-selective intergration of sensory input and vigilance state. S.R. Gray, L. Ye, J.Y Ye, M. Paukert Science Advances 2021; 10.1126/sciadv.abk1378.

39 Publications by other groups using our systemic AAV viruses in FY2021 – 5 highlighted

• An amygdala circuit that suppresses social engagement. J.T. Kwon, C. Ryu, H. Lee, A. Scheffield, J. Fan, D.H. Cho, S. Bigler, H.A. Sullivan, H.K. Choe, I.R. Wickersham, M. Heiman, G.B. Choi Nature 2021; 10.1038/s41586-021-03413-6.

• Chemico-genetic discovery of astrocytic control of inhibition in vivo. T. Takano, J.T. Wallace, K.T. Baldwin, A.M. Purkey, A. Uezu, J.T. Courtland, E.J. Soderblom, T. Simogori, P.F. Maness, C. Eroglu, S.H. Soderling Nature 2020; 10.1038/s41586-020-2926-0.

• Light microscopy based approach for mapping connectivity with molecular specificity. F.Y. Shen, M.M. Harrington, L.A. Walker, H.P.J. Cheng, E.S. Boyden, D. Cai Nature Communications 2020; 10.1038/s41467-020-18422-8.

• Importin alpha3 regulates chronic pain pathways in peripheral sensory neurons. L. Marvaldi, N. Panayotis, S. Alber, S.Y. Dagan, N. Okladnikov, I. Koppel, A. Di Pizio, D.A. Song, Y. Tzur, M. Terenzio, I. Rishal, D. Gordon, F. Rother, E. Hartmann, M. Bader, M. Fainzilber Science 2020; 10.1126/science.aaz5875.

• Astroglial FMRP decificiency cell-autonomously up-regulates miR-128 and disrupts developmental astroglial mGluR5 signaling. Y. Men, L. Ye, R.D. Risgaard, V. Promes, X. Zhao, M. Paukert, Y. Yang Proceedings of the National Academies of Sciences USA 2020; 10.1073/pnas.2014080117

43 Publications by other groups using our systemic AAV viruses in FY2020 – 5 highlighted

• Structure and flexibility in cortical representations of odour space. S.L. Pashkovski, G. Iurilli, D. Brann, D. Chicharro, K. Drummey, K. Franks, S. Panzeri, S. Robert Datta Nature 2020; 10.1038/s41586-020-2451-1

• Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice. H. Zhao, J. Su, X. Hu, C. Zhou, H. Li, Z. Chen, Q. Xiao, B. Wang, W. Wu, Y. Sun, Y. Zhou, C. Tang, F. Liu, L. Wang, C. Feng, M. Liu, S. Li, Y. Zhang, H. Xu, H. Yao, L. Shi, H. Yang Cell 2020; 10.1016/j.cell.2020.03.024.

• Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration. R. Pinto-Costa, S.C. Sousa, S.C. Leite, J. Nogueira-Rodrigues, T. Ferreira da Silva, D. Machado, J. Marques, A. Catarina Costa, M.A. Liz, F. Bartolini, P. Brites, M. Costell, R. Fassler, M.M. Sousa J Clinical Investigation 2020; 10.1172/JCI125771.

• Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. J.M. Levy, W. Yeh, N. Pendse, J.R. Davis, E. Hennessey, R. Butcher, L.W. Koblan, J. Comander, Q. Liu, D.R. Liu Nature Biomedical Engineering 2020; 10.1038/s41551-019-0501-5.

• Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain. J. Winnubst, E. Bas, T.A. Ferreira, Z. Wu, M.N. Economo, P. Edson, B.J. Arthur, C. Bruns, K. Rokicki, D. Schauder, D.J. Olbris, S.D. Murphy, D.G. Ackerman, C. Arshadi, P. Baldwin, R. Blake, A. Elsayed, M. Hasan, D. Ramirez, B. Dos Santos, M. Weldon, A. Zafar, J.T. Dudman, C.R. Gerfen, A.W. Hantman, W. Korff, S.M. Sternson, N. Spruston, K. Svoboda, J. Chandrashekar Cell 2019; 10.1016/j.cell.2019.07.042.

15 Publications by other groups using our systemic AAV viruses in FY2019 – 5 highlighted

• Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector. Rincon, M. Y., de Vin, F., Duque, S. I., Fripont, S., Castaldo, S. A., Bouhuijzen-Wenger, J., Holt, M. G. Gene Therapy, 2018; 10.1038/s41434-018-0005-z

• Gene transfer with AAV9-PHP.B rescues hearing in a mouse model of Usher Syndrome 3A and transduces hair cells in a non-human primate. Gyorgy, B., Meijer, E. J., Ivanchenko, M. V., Tenneson, K., Emon, F., Hanlon, K. S., Indzhykulian, A. A., Volak, A., Karavitaki, K. D., Tamvakologos, P. I, Vezina, M., Merezovskii, V. K., Born, R. T., O'Brien, M., Lafond, J. F., Arsenijevic, Y., Kenna, M. A., Maguire, C. A., Corey, D. P. Molecular Therapy. Methods & Clinical Development, 2018; 10.1016/j.omtm.2018.11.003

• Intravenous injection of an AAV-PHP.B vector encoding human acid a-Glucosidase rescues both muscle and CNS defects in murine Pompe Disease. Lim, J. A., yi, H., Gao, F., Raven, N., Kishnani, P. S., Sun, B. Molecular Therapy. Methods & Clinical Development, 2019; 10.1016/j.omtm.2019.01.006

• The GPI-linked protein LY6A drives AAV-PHP.B transport across the blood-brain barrier. Hordeaux, J., Yuan, Y., Clark, P. M., Wang, Q., Martino, R. A., Sims, J. J., Bell, P., Raymond, A., Stanford, W. L., Wilson, J. M. Molecular Therapy, 2019; 10.1016/j/ymthe.2019.02.013

• Targeted transgene activation in the brain tissue by systemic delivery of engineered AAV1 expressing CRISPRa. Lau, C. H., Ho, J. W., Lo, P. K., Tin, C. Molecular Therapy. Nucleic Acids, 2019; 10.1016/j/omtn.2019.04.015