Jamie M. Bergen

Nonviral Approaches for Neuronal Delivery of Nucleic Acids

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.

Application of an HIV gp41-derived peptide for enhanced intracellular trafficking of synthetic gene and siRNA delivery vehicles.

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.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.

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.

Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons.

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.

Cystine-knot peptides: emerging tools for cancer imaging and therapy

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature’s repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

Peptide-modified vectors for nucleic acid delivery to neurons.

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.

Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration

Significance The delivery of drugs into the central nervous system is limited by the blood–brain barrier. Consequently, there exists an unmet need to develop technologies with the ability to deliver biologic drugs (such as peptides, proteins, or nucleic acids) into the brain or spinal cord via peripheral administration routes. In this paper, a screen of recombinant bacteriophage identified a peptide, targeted axonal import (TAxI), that is able to deliver protein cargo into spinal cord motor neurons after intramuscular injection by a retrograde transport mechanism. The demonstration of a nonviral-mediated delivery system demonstrates the clinical potential of this technology to develop powerful therapeutic tools to treat motor neuron diseases. A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.