Improving efficacy of therapeutics by enhancing delivery using chemical engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2018.Cataloged from PDF version of thesis.Includes bibliographical references.In the past decades, many new and interesting modalities for therapeutics have been discovered, including nucleic acid therapeutics such as siRNA and mRNA. However, one of the limiting challenges in developing these technologies into medicines is delivering the therapeutics to the correct location in the body or in the cell. Furthermore, many older modalities for therapeutics, such as vaccines and chemotherapeutics, could become more efficacious with optimization of delivery. By using chemical engineering principles, we can develop better delivery methods, materials, and formulations to improve the treatment of a wide range of diseases. In this thesis, I report on applications to vaccines and cancer. Vaccines are currently the vanguard of public health efforts; unfortunately, a wide range of diseases have no effective vaccine. This includes devastating diseases such as HIV, malaria, and others. One area of vaccination that few people have considered optimizing is the kinetics by which the vaccine is delivered. We found that using an exponential increasing dosing profile, we could produce over 7 times more antibodies compared to the current prime-boost profile using the same amount and type of vaccine. The antibodies generated were also of higher affinity. By improving antibody affinity and titer, this work may make existing vaccines for diseases such as HIV sufficiently efficacious to use in humans. Cancer is one of the leading causes of death in both developed and developing countries, and is extremely difficult to cure due to its high variability. Furthermore, current cancer therapeutics cause severe toxicity. By delivering more of the cancer therapeutics to the tumor, we can reduce the side effects. Some tumors, because of their location, are even harder to access: brain tumors, such as glioblastoma, are protected from most drugs by the blood-brain barrier or blood-brain-tumor barrier. Circumventing these challenges allow us to develop safer and more efficacious therapies. We found that conjugates of siRNA with chlorotoxin could knock down levels of a housekeeping gene in vitro and in vivo in a mouse brain tumor model. Furthermore, we developed prostate-cancer targeting ligands that demonstrate in vitro efficacy and tested them in vivo.by Hok Hei Tam.Ph. D

Towards accurate calculations of Zn2+ binding free energies in zinc finger proteins

Zinc fingers are Zn2+-bound peptide motifs that bind DNA specifically and have great potential in gene therapy. However, the ion binding strength of the zinc finger is not well known, and computing this quantity will allow for the design of more stable zinc finger treatments. Ions in solution are a model system. Molecular dynamics (MD) simulations and the inverse potential distribution theorem were used to estimate the solvation free energies of zinc ions. The zinc coordination shells were stable and the initial coordination shell stayed throughout the 20 ns simulations. Quasi-chemical (QC) calculations are free energy calculations that partition the system into an inner shell, treated using quantum mechanics, and an outer shell, treated using continuum electrostatics. The theory was extended to multiple ligands in solution and used on Zn2+ in water/methanol mixtures, with the inner shell consisting of the six solvent molecules coordinated to the ion and the outer shell consisting of all other solvent. Increasing methanol coordinated to the zinc led to lower inner shell formation free energies but higher outer shell free solvation energies. A six-water coordination shell was found to be most stable. Using quasi-chemical theory with different concentrations in the outer shell did not yield major differences, but this could have been due to an insufficient treatment of the van der Waals forces. A quasi-chemical approximation using MD to treat the outer shell would fix such problems and will be useful in computing zinc finger ion binding free energies.No embarg

Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination

Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1–2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.National Institute of Allergy and Infectious Diseases (U.S.) (Awards UM1AI100663)National Institute of Allergy and Infectious Diseases (U.S.) (Awards AI110657

Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response.

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices

Long term Glycemic Control Using Polymer Encapsulated, Human Stem-Cell Derived β-cells in Immune Competent mice

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in diabetic patients1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but are limited by the adverse effects of lifetime immunosuppression and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose responsive mature β-cells derived from human embryonic stem cells; called SC-β, these cells may represent an unlimited human cell source for pancreas replacement therapy4. Strategies to address the immunosuppression concern include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier5,6. However, clinical implementation has been challenging due to host immune responses to implant materials7. Here, we report the first long term glycemic correction of a diabetic, immune-competent animal model with human SC-β cells. SC-β cells were encapsulated with alginate-derivatives capable of mitigating foreign body responses in vivo, and implanted into the intraperitoneal (IP) space of streptozotocin-treated (STZ) C57BL/6J mice. These implants induced glycemic correction until removal at 174 days without any immunosuppression. Human C-peptide concentrations and in vivo glucose responsiveness demonstrate therapeutically relevant glycemic control. Implants retrieved after 174 days contained viable insulin-producing cells

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.Leona M. and Harry B. Helmsley Charitable Trust (3-SRA-2014-285-M-R)United States. National Institutes of Health (EB000244)United States. National Institutes of Health (EB000351)United States. National Institutes of Health (DE013023)United States. National Institutes of Health (CA151884)United States. National Institutes of Health (P41EB015871-27)National Cancer Institute (U.S.) (P30-CA14051

Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model.

Klebsiella pneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD50<100 CFU) while MGH 78578 is relatively avirulent

Probit analysis of IMIT-infections of BALB/c mouse using <i>K. pneumoniae</i> strains.

<p>*95% confidence interval.</p><p>Probit analysis of IMIT-infections of BALB/c mouse using <i>K. pneumoniae</i> strains.</p

Primers used in this study.

<p>Primers used in this study.</p