Viviana Gradinaru

Howard Hughes Medical Institute Investigator (selected 2024)
Troendle Professor of Neuroscience and Biological Engineering
Director and Davis Leadership Chair, Merkin Institute for Translational Research
Director, Center for Molecular and Cellular Neuroscience of the Chen Institute
Principal Investigator, Gradinaru Research Laboratory
Faculty Advisor, Beckman Institute CLOVER Center for Neurotechnologies

Professor Gradinaru is a physicist-turned-neuroscientist and engineer of proteins and viruses, motivated by a desire to understand the brain and to act upon that understanding to improve human health. As a neurotechnologist with >15 years of experience, Prof. Gradinaru and her group have developed methods that enable functional and anatomical access to the vertebrate nervous system, such as tools for readout and control of neural activity, tissue clearing and imaging, and gene-delivery vectors. They have used learnings from distinct fields (neuroscience, protein engineering, and data science) to overcome some of the biggest challenges in optogenetics and gene delivery, developing microbial opsins that are tolerated by mammalian cells (e.g. eNpHR3.0, highly used worldwide) and viral capsids capable of crossing the blood–brain barrier in adult mammals (e.g. AAV-PHP.eB, now used by hundreds of groups worldwide and growing). Her group has disseminated new opsins, viruses, and protocols for gene delivery and tissue clearing into the research community (corresponding author on: Treweek et al, Nature Protocols, 2015; Deverman et al, Nature Biotechnology, 2016; Chan et al, Nature Neuroscience, 2017; Challis et al, Nature Protocols, 2019; Bedbrook et al, Nature Methods, 2019; Kumar et al, Nature Methods, 2020). They have also used many of these novel technologies to better understand circuits underlying locomotion, reward, and sleep (Gradinaru et al, Science, 2009; Xiao et al, Neuron, 2016; Cho et al, Neuron, 2017; Oikonomou et al, Neuron, 2019; Challis et al, Nature Neuroscience, 2020). Going forward, Prof. Gradinaru's goal is to enable high-precision, minimally-invasive study and repair of diseased nervous systems across species, by developing a deep mechanistic understanding of viral capsids and the rules governing transport across the blood–brain barrier, and leveraging this knowledge to engineer ideal vehicles for gene delivery to the brain via the vasculature.

Prof. Gradinaru is very active in training and service, notably as faculty director of the Beckman Institute CLOVER (CLARITY, Optogenetics and Vector Engineering Resource) Center, which provides training and access to the group's reagents and methods. They promptly disseminate their tools/reagents and know-how to the broader community for use, improvement, and scientific discovery (Addgene Blue Flame Award 4x; training workshops at Stanford, CSHL, U. Michigan, and Caltech; >100 publications, ~30k citations, h-index 65).

Prof. Gradinaru has received many honors and awards, including the NIH Director's Innovator and Pioneer Awards, the Presidential Early Career Award for Scientists and Engineers, outstanding young investigator awards from the American Society of Gene and Cell Therapy and the Society for Neuroscience, the Innovators in Science Award in Neuroscience from Takeda and the New York Academy of Sciences, the Gill Transformative Investigator Award, the Science Magazine & PINS Prize for Neuromodulation, the Vilcek Prize for Creative Promise in Biomedical Science, and the Great Immigrants Award from the Carnegie Corporation of New York. Gradinaru is a Sloan Fellow, Pew Scholar, Moore Inventor, Vallee Scholar, World Economic Forum Young Scientist, and a Fellow of both the National Academy of Inventors and the American Association for the Advancement of Science (AAAS).

Major Research Contributions:

(1) Engineered viral vectors for targeted, noninvasive access to the mammalian brain

The ability to introduce genetically-encoded cellular sensors and modifiers—including ones Prof. Gradinaru developed—to the brain is critical in helping us understand circuits responsible for mammalian behavior and pathophysiology. However, conventional gene-delivery methods have drawbacks: transgenesis is slow, and infeasible in many species, and intracranial injections are invasive and provide limited coverage. In theory, one could access the entire brain via the vasculature, but access by macromolecules (including adeno-associated viral vectors–AAVs) is blocked by the formidable blood-brain barrier (BBB). To unlock this gateway, the Gradinaru lab leveraged a fascinating feature of AAVs: residue differences on the capsid surface alter tissue and cell-type tropism. Over the last decade, they developed and refined a platform for generating structural diversity in the capsid and selecting variants that display desired properties, e.g., ability to cross the BBB. In work begun with Dr. Ben Deverman when he was in the group, they realized that the key lay in selectively retrieving successful variants. Using transgenic mouse lines expressing Cre recombinase in target cells, they established in vivo Cre-recombination-based AAV targeted evolution (which Prof. Gradinaru named CREATE, ref. a). They next tackled an even bigger challenge: addressing AAVs to particular cell populations. For this, they designed a multiplexed platform employing multiple Cre lines and a computational pipeline for combinatorial selection of both positive and negative criteria (ref. b). Their work introduced a potent toolkit of systemic AAVs for diverse targets in the brain, including neurons, astrocytes, and vasculature (refs. c-d), with near-complete de-targeting of peripheral organs including liver, heart, and dorsal root ganglia. These tools for gene delivery to the rodent brain have been cited ~2,000 times and are used by hundreds of labs, including Prof. Gradinaru's, for basic and preclinical research ranging from previously-impossible brain-wide genetic screens to transforming the BBB into an in vivo therapeutic biofactory (Contribution 3 ref. c).

(2) Tools for precise optical modulation and readout of neuronal activity in situ

The early development of optogenetics presented a number of challenges, which Prof. Gradinaru's work helped solve. Many opsins, especially pumps, were not well tolerated by mammalian cells, so she worked out cellular trafficking strategies that resulted in potent and safe optogenetic tools (e.g., eNpHR), including inhibitors that span the visible spectrum, and generalizable strategies for targeting cells based on genetic identity or projection patterns. To overcome poor tissue penetration by visible light, in collaboration with Prof. Frances Arnold at Caltech, her group used directed evolution and machine learning to develop high-fluxing opsins that allow activation by a distant light source, which, in combination with systemic viral delivery, brings minimally-invasive brain modulation closer to reality (ref. a). Traditionally, neural circuits were mapped by sectioning tissue, imaging each slice, and reconstructing them with software, a tedious and error-prone process. As an alternative, Prof. Gradinaru contributed, while working with Karl Deisseroth, to a method known as CLARITY, which removes view-obstructing lipids and renders the tissue transparent for imaging without slicing. Expanding the utility of CLARITY, the Gradinaru lab reported the first whole-body clearing via vasculature perfusion, producing transparent rodents for mapping central and peripheral nerves (ref. b). These methods can also reveal the position/impact of electrical or optical probes in situ within circuits of interest (published as LiGS). Opsins can be engineered for diverse properties, including optical readout of membrane voltage changes. They used directed evolution of opsins to make them better at reporting action potentials (ref. c). Preserving spatial relationships while accessing the transcriptome of selected cells is crucial for advancing many areas of biology, from developmental biology to neuroscience, where the activity history of brain circuits is recorded in RNA transcripts. Collaborating with Profs. Long Cai and Niles Pierce at Caltech, they developed methods for multi-color, multi-RNA imaging in deep tissues. Going further, they combined signal amplification and tissue clearing to develop an ultrasensitive sequential FISH (USeqFISH) method for multiplexed detection of up to 50 genes at a time in 50-µm-thick tissue slices (ref. d), enabling high-throughput and high-resolution spatial transcriptomics in situ, which is critically important for tissues with heterogeneous microarchitecture like the brain.

(3) Opening blood–brain barrier portals for molecular delivery in biologically diverse organisms

As described in Contribution 1, the Gradinaru lab now has a strong toolkit of AAVs for non-invasive gene delivery to the rodent brain. Disappointingly, most of them fail to cross the BBB in non-human primates (NHPs). Figuring that the best chance for translating transduction properties to humans lay in getting as evolutionarily close as possible, meaning Old World monkeys, and wanting to maximize this precious resource, they developed a multi-species directed evolution strategy, performing two selection rounds in adult marmosets (a New World monkey) before a final selection round in infant rhesus macaques (Old World). Using this approach, they identified a promising new family of BBB-crossing AAVs and validated the archetype AAV.CAP-Mac in two Old World monkey species (ref. a). In both, CAP-Mac broadly transduced neuronal subtypes throughout the brain, enabling exciting functional and anatomical paradigms, including unprecedented macaque whole-brain Brainbow labeling to characterize neuronal morphology. They also used CAP-Mac to demonstrate the first non-invasive brain-wide delivery of GCaMP to an Old World primate. In separate work (refs. b and c), they engineered additional AAVs from distinct sequence families that can efficiently cross the macaque and marmoset BBB and display diversified tropism once across the barrier. This vector suite, which outperforms existing tools in cultured macaque and human brain slices, offers an unprecedented opportunity to apply powerful neurotechnology to the non-transgenic primate brain. As Prof. Gradinaru's group pioneered discovery of BBB-crossing AAVs in various species, others in academia and industry adopted and adapted their in vivo directed-evolution methods, building a rich toolbox of systemic AAVs. A curious feature of this toolbox is that the mechanism-agnostic nature of the engineering means we know nearly nothing about how these AAVs (1) cross the BBB and (2) access distinct cell populations in the brain. Building a platform to combine structural information with in vitro and in silico methods, they discovered two novel BBB receptors for AAVs (ref. d): carbonic anhydrase IV (CA4), which is widely conserved across mammals, and the murine-restricted Ly6c. Particularly exciting was CA4, an enzyme on the endothelial cell surface that mediates the balance of carbon dioxide and bicarbonate in the vasculature, an unexpected player in BBB transcytosis. In addition to uncovering new biology, these receptors offer attractive targets for engineering, providing a new path toward efficient human-brain-penetrant chemical and biological therapeutics.

(4) Expanded understanding of key central and peripheral nervous systems circuits underlying neurological and neuropsychiatric disorders

The tools and methods the Gradinaru group developed and disseminated have enabled thousands of labs to advance our understanding of biological circuits underlying, among many things, digestion, cardiac physiology, pain, neurodevelopment, neurodegeneration, and behavior. Their own work has largely focused on circuits underlying neurodegenerative disorders, beginning with Gradinaru's PhD study of optogenetic deep-brain stimulation for the treatment of Parkinson's disease (PD; Gradinaru et al., Science 2009). Recently, she has become fascinated by the broader context of brain-body interactions, for instance how neuronal dynamics in the brain's circadian pacemaker control ovulation (ref. a) or how physiological state feeds back on the brain to modulate motivation (ref. b) or drive sleep (ref. c). Recent evidence suggests that in PD, pathological α-Synuclein (αSyn) aggregation begins in peripheral tissues and progresses to the brain. Her group tested this by inoculating the mouse duodenum with pre-formed αSyn fibrils. Using new AAV tools they developed for the peripheral nervous system, they observed physiological changes to the enteric nervous system that were followed by pathology in the brain and motor symptoms. Importantly, they did not see the same effects in younger mice. They also found that peripheral αSyn pathology was reduced by increased expression of the lysosomal enzyme glucocerebrosidase, defects in which are associated with PD and Gaucher disease. This work (ref. d) shifts the focus of neurodegenerative disease etiology from the CNS to the PNS and adds evidence for the "multiple-hit" hypothesis of genetic, environmental, and age factors collaborating in neuropathology. Their findings provide motivation for understanding (and eventually treating) both central and peripheral physiology, while their technical work enables this vision by breaking with the necessity of transgenics, which has severely limited primate and multifactorial physiology studies to date.