(6m) Biomolecular Engineering and Ultrasound-Enhanced Transport in Neuroscience

The Szablowski laboratory will develop technologies for perturbing neural circuits to study brain function and treat neurological and neuropsychiatric disease. We will achieve these goals through a combination of unique engineering strategies aimed at overcoming transport limitations in the brain using focused ultrasound, and allowing specific and efficient control of neuronal cells through biomolecular engineering.

Ultrasound can be used to deliver molecules locally to tissues with millimeter precision, a level of accuracy required to selectively modulate brain circuits. Ultrasound-mediated gene delivery will allow us to achieve spatially selective long-term perturbation of brain circuits in a fully non-invasive manner. We will combine enhanced transport across tissue boundaries with biomolecular engineering. This work will utilize genetic engineering, protein receptor engineering, and next-generation sequencing to develop tools to noninvasively modulate neurons and investigate circuit function. Neuroscience is central to human health and due the complexity of the brain, controlling neural circuits is a perfect testing-ground for creative biomolecular engineering strategies. This research program will establish new engineering principles and yield discoveries in both basic science and translational medicine.

Research experience

Postdoctoral: Ultrasound-Modulated Transport of Genetic Vectors Into the Brain for Non-Invasive Control of Neural Circuits (Advisor: Mikhail G. Shapiro, Chemical Engineering, Caltech, 06/2015-current)

The treatment of neuropsychiatric and neurological diseases, such as mood disorders, or epilepsy, requires control of the right cells within the right brain region at the right time. However, currently, this cannot be achieved noninvasively. For example small molecule therapeutics can act on selected protein receptors, but are delivered non-selectively throughout the brain. On the other hand deep brain stimulation can target a specific region within the brain but requires a surgery and a permanent intracranial placement of an electrode.

During my postdoctoral training I initiated a new area of research to develop a methodology that can be both noninvasive and have regional, cell-type, and temporal specificity of neuromodulation. This technology, called Acoustically Targeted Chemogenetics¹ (in press, Nature Biomedical Engineering), or ATAC, combines three cutting-edge techniques with clinical potential. First, ultrasound is used for gene delivery across blood-brain barrier (BBB). When microscopic vesicles containing gas are infused intravenously focused ultrasound (FUS) stimulates their oscillations which exert weak pressure on the brain vasculature temporarily loosening junctions between endothelial cells thus opening the BBB (BBBO). Consequently, viral vectors that cannot pass through an intact BBB, but readily do so after BBBO, can be selectively delivered to a sonicated brain region. Second, by using those viral vectors to deliver engineered protein receptors that respond to BBB-permeable bio-inert drugs (chemogenetic receptor-ligand pairs), selected neurons can be controlled pharmacologically, which results in noninvasive neuromodulation. And third, by using cell specific promoters a subpopulation of neurons can be targeted with spatial accuracy beyond what is offered by FUS. In this project, we pharmacologically activated and inhibited neurons specifically targeted by ATAC. We then used this technique to inhibit formation of traumatic memories in mice and demonstrated a new method of sub-millimeter accuracy of targeting. Current efforts include further improvements of safety and costs, and translation of ATAC into non-human primates.

In addition to this main project, I contributed to another effort focused on engineering new biomolecular reporters for magnetic resonance imaging² (Nature Materials, 2018), and co-authored two comprehensive reviews on the combination of biomolecules and ultrasound.

Graduate: Molecular Engineering of programmable therapeutics for oncology: drug delivery and mechanism of action (Peter Dervan, Bioengineering, Caltech, 09/2009-05/2015).

Altering gene expression is one of the most attractive therapeutic strategies. Multiple diseases, such as cancer, are a result of improper gene regulation. Additionally, DNA has a simple and predictable structure that is independent of its sequence, which allows similar compounds to target multiple DNA sequences and thus multiple diseases. Consequently, a number of therapeutic approaches focused on DNA have emerged such as CRISPR gene editing, RNA interference, or gene therapy. However all of these methods require gene delivery which is more expensive and less established than use of small molecule therapeutics. The Dervan lab has developed small molecules that are cell-permeable, have pharmacokinetics and biodistribution of small molecules, but can bind to DNA sequence-specifically to regulate gene expression similarly to genetic approaches.

In my graduate work I focused on understanding in vivo selectivity, mechanisms of action, and clinical applicability of DNA-sequence specific small molecule drugs. We evaluated gene expression effects and biodistribution following polyamide treatment in a mouse model of breast cancer and glioma. Using intravital bioluminescence imaging and next generation mRNA sequencing studies, we discovered that one compound targeted to the Estrogen-Response-Element was capable of downregulating estrogenic gene expression in tumors⁵. We also evaluated pharmacokinetics and biodistribution of Py-Im polyamides in both tissues and tumors⁴.

For my next project³, I focused on one of the most critical pathways in cancer – the hypoxic response. Resistance to hypoxia is one of the hallmarks of tumor cells and is one of the major sources of resistance to radiation and chemotherapy. Existing anti-angiogenic therapies, which attempt to starve tumors of their blood supply, can actually make tumors more resistant to hypoxia, thereby promoting tumor progression and metastasis. I attempted to develop an antiangiogenic therapy that does not have this confounding effect. Using a Py-Im polyamide compound targeted to Hypoxia Inducible Factor-1 Response Elements (HRE), I discovered that systemic administration of this compound reduced tumor growth, inhibited blood vessel formation, and sensitized the tumor cells to hypoxia. The Py-Im polyamides are being actively translated to the clinic as cancer therapeutics by Dr. Dervan (through his startup Gene Sciences Inc. in San Diego).

Teaching Interests: My teaching interests include the fundamentals of chemical and biomolecular engineering and an elective focus on therapeutics. Thanks to my undergraduate and graduate training in highly quantitative Bioengineering programs, I have experience with core thermodynamics, transport and kinetics concepts that are critical for Chemical Engineering students. I will leverage my graduate and postdoctoral training and provide students with an ability to intuitively understand how molecules interact and distribute in cells, tissues and organisms. This knowledge will allow them to apply what they learned in core courses to design and engineer new materials and therapeutics. In the future I would like to develop a course on molecular recognition and selectivity in vivo.

Teaching experience:

• Mentoring: 2 undegraduate, 1 graduate student (Caltech, 2016-present)
• Guest lecturer, ChemE 188, Molecular Imaging (Caltech, 2017-present)
• Biology Tutor for Biology Undergraduate Education and Office of Minority Education (MIT, 2007-2008)
• Teaching a seminar course in introductory biology, for Office of Minority Education (MIT, 2008)

Selected publications (out of 12 total)
First-author contributions are underlined

1. Szablowski JO, Lue B, Lee-Gosselin A, Malounda D, Shapiro MG, Acoustically Targeted Chemogenetics for Noninvasive Control of Neural Circuits, in press, Nature Biomed. Eng. (2018)
2. Lu GJ, Farhadi A, Szablowski JO, Barnes SR, Lakshmanan A, Bourdeau RW, Shapiro MG, Acoustomagnetic imaging with gas-filled protein nanostructures, Nature materials 17 (5), 456 (2018)
3. Szablowski JO, Raskatov JA, Dervan PB. An HRE-binding Py-Im polyamide impairs hypoxic signaling in tumors. Mol. Cancer Ther. 15 (4), 608-617 (2016)
4. Raskatov JA, Szablowski JO, Dervan PB, “Tumor Xenograft Uptake of a Py Im Polyamide Varies as a Function of Cell Line Grafted”, J. Med. Chem., 57:8471-8476 (2014)
5. Nickols NG#, Szablowski JO#, Hargrove AE, Li BC, Raskatov JA, Dervan PB. "Activity of a Py-Im Polyamide Targeted to the Estrogen response Element," Mol. Cancer Ther., 12:675-684, (2013).
   # co-first authors, (Article selected as one of the ‘AACR hot topics, 2013’).
6. Shapiro MG#, Westmeyer GG#, Romero P, Szablowski JO, Küster B, Shah A, Otey CR, Langer R, Arnold FH, & Jasanoff AP, “Directed evolution of an MRI contrast agent for noninvasive imaging of dopamine”. Nature Biotech., 28:264–270 (2010), # co-first authors

Patents and patent applications

1. Szablowski JO, Shapiro MG, Acoustically Targeted Chemogenetics, CIT File No.: CIT-7921-P, Provisional filed: 12/7/2017
2. Lu G, Farhadi A, Szablowski JO, Shapiro MG, Gas Filled Structures and related compositions, methods and systems for magnetic resonance imaging, CIT File No.: CIT-7580-P, Provisional filed: 7/28/2016, Patent application filed: 7/28/2017
3. Szablowski JO, User-adjustable knee orthosis for patellar instability and related disorders, CIT File No.: CIT 12-216, Provisional Filed: 8/7/2012