Doctor of Philosophy, University of Washington (2008)
Bachelor of Science, Duke University (2003)
Jennifer Cochran, Postdoctoral Faculty Sponsor
The knottin family offers a diverse array of molecular scaffolds that can be engineered to guide imaging agents and drugs to sites of disease. My research focuses on using knottins as a platform to design targeted drug therapies, and I am studying the delivery characteristics of engineered knottin-drug conjugates in relevant disease models.
Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5β1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.
View details for DOI 10.1073/pnas.1311333110
View details for Web of Science ID 000323886200034
Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.
View details for DOI 10.1371/journal.pone.0030293
View details for Web of Science ID 000301640600024
View details for PubMedID 22295079
We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.
View details for DOI 10.1128/JVI.05154-11
View details for Web of Science ID 000293626100051
View details for PubMedID 21697483
Vascular calcification is highly correlated with morbidity and mortality, and it is often associated with inflammation. Vitamin D may regulate vascular calcification and has been associated with cardiovascular survival benefits.We developed a macrophage/smooth muscle cell (SMC) coculture system and examined the effects of vitamin D receptor activators (VDRA), calcitriol and paricalcitol, on SMC matrix calcification. We found that treatment of SMC alone with VDRA had little effect on phosphate-induced SMC calcification in vitro. However, coculture with macrophages promoted SMC calcification, and this was strikingly inhibited by VDRA treatment. Several VDRA-induced genes, including bone morphogenetic protein-2 (BMP2), tumor necrosis factor-alpha, and osteopontin, were identified as candidate paracrine factors for the protective effect of VDRA. Of these, osteopontin was further investigated and found to contribute significantly to the inhibitory actions of VDRA on calcification in macrophage/SMC cocultures.The ability of VDRA to direct a switch in the paracrine phenotype of macrophages from procalcific to anticalcific may contribute to their observed cardiovascular survival benefits.
View details for DOI 10.1161/ATVBAHA.109.196576
View details for Web of Science ID 000273799900031
View details for PubMedID 19948844
Neuron-targeted nucleic acid delivery systems are important technologies for realizing the potential of gene therapy for nervous system disorders. However, neurons are difficult cells to transfect using non-viral vectors due in part to the specific and unique delivery challenges present in these cells. We have investigated several bioactive peptides for their ability to assist in overcoming delivery barriers in mammalian cells. We summarize here our recent progress in developing and applying peptide-modified polycations for nucleic acid delivery. In addition, we present data demonstrating the potential of using multicomponent, peptide-modified polycations for nucleic acid delivery to neurons.
View details for DOI 10.1016/j.jconrel.2008.06.012
View details for Web of Science ID 000261902800012
View details for PubMedID 18627784
The delivery of therapeutic nucleic acids to neurons has the potential to treat neurological disease and spinal cord injury. While select viral vectors have shown promise as gene carriers to neurons, their potential as therapeutic agents is limited by their toxicity and immunogenicity, their broad tropism, and the cost of large-scale formulation. Nonviral vectors are an attractive alternative in that they offer improved safety profiles compared to viruses, are less expensive to produce, and can be targeted to specific neuronal subpopulations. However, most nonviral vectors suffer from significantly lower transfection efficiencies than neurotropic viruses, severely limiting their utility in neuron-targeted delivery applications. To realize the potential of nonviral delivery technology in neurons, vectors must be designed to overcome a series of extra- and intracellular barriers. In this article, we describe the challenges preventing successful nonviral delivery of nucleic acids to neurons and review strategies aimed at overcoming these challenges.
View details for DOI 10.1007/s11095-007-9439-5
View details for Web of Science ID 000254849700001
View details for PubMedID 17932730
Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.
View details for DOI 10.1021/bc700448h
View details for Web of Science ID 000255068000015
View details for PubMedID 18376855
Neuron-specific, nonviral gene delivery vehicles are useful tools for the potential treatment of neurological disease and spinal cord injury. For minimally invasive, peripheral administration, gene carriers must efficiently mediate uptake at axon terminals, retrograde axonal transport, vesicular escape, and nuclear entry. The design of improved vehicles will benefit from an understanding of the barriers that limit nonviral delivery to neurons. Here, we demonstrate a detailed analysis of intracellular trafficking of both a lipid-based and a polymer-based delivery vehicle following site-specific exposure to neuron-like cells.Site-specific exposure of gene carriers to soma or neurites of neuron-like PC-12 cells was accomplished using a microfluidic, compartmented culture chamber. Binding and internalization of vehicles at neurites and soma were quantified using an environmentally sensitive fluorescent marker. The intracellular transport of gene carriers was analyzed by time-lapse particle tracking in live cells, and transfection efficiencies were measured using green fluorescent protein (GFP) as a reporter gene.While the lipid-based carrier mediated measurable transfection when delivered to neuronal soma, neuritic delivery of this formulation failed to produce reporter gene expression due to limited internalization and transport. In contrast, the polymeric nanoparticles displayed active retrograde transport toward neuronal soma, but failed to produce measurable reporter gene expression.These results highlight distinct intracellular barriers preventing efficient neuronal transfection by the nonviral carriers examined, and provide a basis for the rational improvement of existing nonviral systems.
View details for DOI 10.1002/jgm.1137
View details for Web of Science ID 000253430100008
View details for PubMedID 18064730
Nonviral gene carriers must associate with and become internalized by cells in order to mediate efficient transfection. Methods to quantitatively measure and distinguish between cell association and internalization of delivery vectors are necessary to characterize the trafficking of vector formulations. Here, we demonstrate the utility of nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled oligonucleotides for discrimination between bound and internalized gene carriers associated with cells. Dithionite quenches the fluorescence of extracellular NBD-labeled material, but is unable to penetrate the cell membrane and quench internalized material. We have verified that dithionite-mediated quenching of extracellular materials occurs in both polymer- and lipid-based gene delivery systems incorporating NBD-labeled oligonucleotides. By exploiting this property, the efficiencies of cellular binding and internalization of lipid- and polymer-based vectors were studied and correlated to their transfection efficiencies. Additionally, spatiotemporal information regarding binding and internalization of NBD-labeled gene carriers can be obtained using conventional wide-field fluorescence microscopy, since dithionite-mediated quenching of extracellular materials reveals the intracellular distribution of gene carriers without the need for optical sectioning. Hence, incorporation of environmentally sensitive NBD-oligos into gene carriers allows for facile assessment of binding and internalization efficiencies of vectors in live cells.
View details for DOI 10.1021/bc700315v
View details for Web of Science ID 000252520300048
View details for PubMedID 18062659
The limited cytoplasmic mobility of nonviral gene carriers is likely to contribute to their low transfection efficiency. This limitation could be overcome by mimicking the viral strategy of recruiting the dynein motor complex for efficient transport toward the host cell nucleus. A promising approach for attaching artificial cargo to dynein is through an adaptor peptide that binds the 8 kDa light chain (LC8) found in the cargo-binding region of the dynein complex. Several viral proteins that bind LC8 have in common an LC8-binding motif defined by (K/R)XTQT. Short peptides containing this motif have also been shown to bind recombinant LC8 in vitro. However, since the majority of intracellular LC8 exists outside of the dynein complex, it remains unclear whether peptides displaying this LC8-binding motif can access and bind to dynein-associated LC8. In this study, we employed biochemical analysis to investigate the feasibility of attaching artificial cargo to the dynein motor complex using a peptide displaying the well-characterized LC8-binding motif. We report that free intracellular LC8 bound specifically to an LC8-binding (TQT) peptide and not to a control peptide with a mutated LC8-binding motif. However, a similar binding interaction between the TQT peptide and intracellular dynein was not detected. To determine whether dynein binding of the TQT peptide was prevented by competition with free intracellular LC8 or due to the inability of the peptide to access its LC8 binding site in the dynein complex, the TQT peptide was evaluated for its ability to bind either purified LC8 or purified dynein. Our results demonstrate that, while the TQT peptide readily binds free LC8, it cannot bind to dynein-associated LC8. The results emphasize the need to identify functional dynein-binding peptides and highlight the importance of designing peptides that bind to the intact dynein motor complex.
View details for DOI 10.1021/mp060086o
View details for Web of Science ID 000247241800011
View details for PubMedID 17274669
The development of targeted vehicles for systemic drug delivery relies on optimizing both the cell-targeting ligand and the physicochemical characteristics of the nanoparticle carrier. A versatile platform based on modification of gold nanoparticles with thiolated polymers is presented in which design parameters can be varied independently and systematically. Nanoparticle formulations of varying particle size, surface charge, surface hydrophilicity, and galactose ligand density were prepared by conjugation of PEG-thiol and galactose-PEG-thiol to gold colloids. This platform was applied to screen for nanoparticle formulations that demonstrate hepatocyte-targeted delivery in vivo. Nanoparticle size and the presence of galactose ligands were found to significantly impact the targeting efficiency. Thus, this platform can be readily applied to determine design parameters for targeted drug delivery systems.Modified gold nanoparticles are a suitable model for nanoparticle-based gene carriers.
View details for DOI 10.1002/mabi.200600075
View details for Web of Science ID 000239399100004
View details for PubMedID 16921538
View details for Web of Science ID 000231996500019