Polyplex transfection from intracerebroventricular delivery is not significantly affected by traumatic brain injury

Highlights

• Polymer-mediated gene transfer efficiency to the brain is not inhibited by injury.

• Maximal VIPER mediated gene delivery occurs in the brain three days post-injury.

• CNS glycosaminoglycans are a likely barrier to polyplex-mediated gene delivery.

Abstract

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negatively charged NG2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo.

Introduction

Approximately 1.7 million people suffer a traumatic brain injury (TBI) each year in the United States alone, injuries that can result in death or permanent disability while costing the healthcare system over $50 billion annually. [1,2] While focal TBI is initiated as an acute blunt-force trauma to the exterior of the skull that causes immediate cortical zone cell death, larger ischemic or swollen regions then develop throughout the cortex that produce permanent scarring and neuron loss [3]. The current standard of care for TBI seeks to curtail these secondary damages by limiting bleeding and inflammation but requires prompt implementation and does not ultimately restore the function of the neural tissue lost to injury [4]. This is in part because the injured central nervous system (CNS) has limited endogenous neurogenic capacity and the injury environment negatively impacts neural precursor migration, differentiation and functional integration [5]. Thus, therapeutic strategies for the regeneration and integration of neurons at the site of injury could greatly impact patient recovery after TBI.

While stem cell transplantation therapies for tissue regeneration in the CNS have rapidly progressed into clinical study [6], gene therapies that manipulate endogenous neural progenitor cells (NPCs) with transcription or growth factors offer a promising alternative [[7], [8], [9]]. Compared to cultured cell therapies used as transplants, “direct reprogramming” approaches are more cost effective, less toxic, and promise access to different neuronal subtypes that may repair damaged nerve circuits with greater efficacy [8,10]. Interestingly, it has been shown that a variety of injuries including TBI stimulate the endogenous reservoir of NPCs in the subventricular zone (SVZ) to proliferate and migrate to the site of injury in adult rodents, primates, and humans [[11], [12], [13], [14]]. Upon arrival in the TBI cortex, these cells primarily differentiate into astrocytes but also a small number of neurons, resulting in minor improvements to motor and sensory function in rodents [15,16]. While naturally-occurring neurogenesis is not enough to restore function following TBI, it motivates research that will enhance the proliferation, migration, and differentiation of SVZ NPCs into cortical neurons [8,10]. Although direct injection of transcription factor-expressing viruses into the injured CNS has shown promise as a means for reprogramming endogenous cells into neurons [[17], [18], [19], [20]], viral strategies are translationally limited by their immunogenicity, genetic cargo capacity, and complex manufacturing processes in comparison to non-viral methods [21,22]. Thus, we seek to augment the NPC repair response through non-viral transfection of therapeutic genes in SVZ NPCs through intracerebroventricular (ICV) injection of polyplexes.

We have previously demonstrated delivery of reporter plasmids to cells of the SVZ in healthy mice [23,24]; however, very little is known about the impact of TBI on in vivo transfection. In this work, we utilize our most promising polymeric gene carrier (VIPER [24,25], Scheme 1) to deliver various plasmid cargoes through ICV injection in a controlled cortical impact (CCI) mouse model of TBI. Although the clinical etiology of TBI is extremely diverse, CCI offers promise as a translational model that generates reproducible and quantifiable cognitive and motor deficits in mice that mimic human symptoms [26,27]. As a first step towards therapeutic transfection after TBI, we first optimize the timing of transfection post-injury using luciferase reporter plasmids in order to capitalize on the dynamic cellular proliferation response to injury. We next analyze the distribution of GFP transfection after injury in various brain regions contacting the ventricular space through confocal microscopy, and then further investigate extracellular barriers to gene delivery in these regions through a combined in vivo and in vitro approach. This work highlights the significant hurdles between current non-viral transfection approaches and therapeutic transfection after CCI in mice and establishes guidelines for future vector development.