Title: Gene therapy and nerve repair

Cover Page

The handle http://hdl.handle.net/1887/43807 holds various files of this Leiden University dissertation

Author: Hoyng, Stefan

Title: Gene therapy and nerve repair

Issue Date: 2016-11-01

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OUTLINE

In the introduction (Chapter 1), we discuss the potential of gene therapy in the context of peripheral nerve injury and surgical repair. We provide an overview of: 1) the main scientific challenges in the field of peripheral nerve repair, 2) the potential of gene ther- apy to enhance nerve regeneration, and we 3) discuss the results of the first animal stud- ies in which gene therapy and surgical repair have been combined. Finally, the future challenges of translating these results to the clinic are discussed and we present four possible future clinical scenarios.

In Chapter 2, our goal was to investigate whether the current standard in nerve surgery (autografting) could be further improved through the overexpression of neurotrophic factors using gene therapy. We performed a large in-vivo comparison by overexpress- ing six neurotrophic factors in a rat model of nerve autografting. We assessed early (2) and late (20 weeks) effects performing histological, electrophysiological and functional analysis. Three of the six neurotrophic factors investigated (BDNF, GDNF and NGF) showed promising early stimulatory effects on axon growth but persistent expression revealed selective but excessive sensory (NGF) or motor axon (BDNF, GDNF) growth and uncontrolled cell proliferation. This resulted in trapping of regenerating axons in the autograft which interfered with recovery of function. These results highlight the neces- sity to develop new tools permitting regulated gene expression thus allowing temporary beneficial expression while avoiding prolonged negative effects resulting from uncon- trolled expression.

In Chapter 3, our objective was to reduce misdirection of regenerating axons by attempt- ing to direct regenerating motor axons through the overexpression of a neurotrophic factor (GDNF) in a single branch of a nerve bifurcation model. We assessed axon rout- ing at 4 weeks by performing retrograde tracing and histology. Tracing and qualitative analysis of the histology showed a trend towards successful routing of motor axons. As in the previous chapter we observed trapping of axons distal to the bifurcation because of uncontrolled expression. This proof of concept study shows that gene therapy may be applied in the future to direct regenerating axons. This will however, only be possible if tight control of neurotrophic expression can be achieved.

In Chapter 4, our aim was to develop a viral vector system that allows regulated expres- sion of a neurotrophic factor in the peripheral nerve. We based our vector on the clas- sical tetracycline-dependent transactivator system. In this so called “Tet-on” system, in-vivo transgene expression can be regulated by the oral administration of doxy cycline (a tetra cycline analog agonist). One of the major drawbacks of this system is that it elicits a rapid immune response directed against one of its essential components: the trans- activator (rtTA). In this chapter we present a regulatable viral vector based on a novel

Proefschrift_Stefan_Hoyng.indd 9 22-9-2016 12:02:48

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transactivator (GArrtTA) potentially capable of evading the immune system. More importantly we show its long-term capability to successfully regulate neurotrophic fac- tor expression in the peripheral nerve.

Our last experimental chapter sets out to investigate alternative viral vectors capable of transducing the peripheral nervous system. Gene therapy for the peripheral nervous system is mainly based on lentiviral vectors. These vectors are very efficient but insert their genetic material into the host cell genome. This random insertion can potentially lead to the activation or inactivation of genes that control cell proliferation leading to cancer. In Chapter 5, we investigated whether a safe, reliable and clinically acceptable viral vector, the adeno-associated viral vector (AAV), was capable of transducing cul- tured rat and human peripheral nerve Schwann cells as well as nerve tissue segments.

We compared the transduction efficiency of 9 AAV serotypes. The data show that rat and human Schwann cells and nerve segments are efficiently transduced by entirely dif- ferent AAV serotypes. The differential transduction efficiency of AAV-serotype vectors for rat and human tissue highlights one of the challenges of translating gene therapy from experimental animals to human patients. Interestingly we showed that AAV2 is the most efficient vector to transduce human nerve segments. AAV2 has been used in several clinical trials and has an excellent safety profile. Therefore, AAV2 is currently the leading vector for the genetic modification of human peripheral nerve autografts.

In Chapter 6, we end with a discussion identifying key areas of future research in the domain of PNS-gene therapy and provide a perspective on the path to clinical trans- lation. We discuss the route and mode of delivery of the future viral vector, the efficacy and safety requirements of this vector as well as the choice of patient population for a first possible proof-of-concept clinical study.

Proefschrift_Stefan_Hoyng.indd 10 22-9-2016 12:02:49