Long‐term in vivo transduction of neurons throughout the rat central nervous system using novel helper‐dependent CAV‐2 vectors

Abstract

Numerous genetic and environmental causes, variable pathophysiologies, and the blood-brain barrier create a formidable challenge for the study and treatment of neurodegenerative diseases affecting the central nervous system. Although there are many intracellular strategies to address neurodegeneration, for example, which transgene to use, one fundamental criterion for the long-term survival of neurons may be their genetic modification. Here, we describe the generation and in vivo efficacy of helper-dependent canine adenovirus (CAV-2) vectors that preferentially transduced neurons and efficiently trafficked via axonal retrograde transport. We used a flexible strategy and the synergy between Cre/loxP and nonlethal packaging-defective helper vectors to generate high titer helper-dependent vector stocks. One year after striatal injections in the rat brain, we found stable, high-level expression in striatal neurons, ~50% of the dopaminergic neurons of the substantia nigra, and the cholinergic neurons in the basal nuclei of Meynert. Due to the intrinsic properties of helper-dependent CAV-2 vectors (27-kb cloning capacity; low preexisting, innate, and induced immunogenicity; retrograde transport; and long-term transgene expression), they will aid fundamental and applied studies in neurobiology. Moreover, helper-dependent CAV-2 vectors may be clinically relevant for the treatment of many neurodegenerative diseases.

Gutless adenovirus: last-generation adenovirus for gene therapy

“…109 On the contrary, gutless adenovirus expression can be detected in the CNS 1 year after striatal injections. 20,110 Moreover and contrary to what was reported for other tissues, preimmunization with the same serotype of adenovirus does not alter gutless stability in the CNS, and does not significantly reduce the initial infection of the CNS by gutless vectors, although a transient acute brain inflammation is shown. 75 Gutless vectors have also been used to successfully express therapeutic genes in the retina either by transplanting cells previously transduced with the virus or by direct injection of the vector.…”

“…However, since both helper and gutless vectors have the same viral capsid, separation must be addressed before purification. Thus far, strategies have been based on reducing the packaging efficiency of the helper genome compared to the gutless genome, either by mutating its packaging signal, [18][19][20] by the different 13 (genomes bigger or smaller than the optimal do not package efficiently), or by specific elimination of its packaging signal during viral production. 21 Initial strategies consisted in the use of helperdependent adenoviruses carrying a defective packaging signal.…”

“…58 Thus, administration of gutless CAV-2 vectors allows the stable, high-level expression in neurons throughout the rat central nervous system (CNS). 20 It is noteworthy that even in the presence of an active peripheral immunization with an adenovirus that completely eliminates expression from first-generation vectors within 60 days, gutless vectors are able to maintain a long-term transgene expression in the brain 74,75 due to reduced inflammation both in intensity and in duration in the brains of the preimmunized animals.…”

“…110,111 Other tissues like lung, cardiac muscle, vascular tissue or dendritic cells have also been targeted with gutless adenoviruses with similar results as for the muscle or the liver. [112][113][114][115] Future considerations This is a fascinating moment for gutless adenovirus vectors: their use permits long-term expression in vivo with reduced and transient cellular immune response in Gutless adenovirus R Alba et al animal models for human diseases and, moreover, the increasing number of groups working in this field favors technological advances to escape preimmune response by developing non-human gutless adenovirus 20 or to increase stability by generating integrative gutless vectors, 116,117 or vectors with replication capacity. 118 However, their use in clinical assays is questionable since helper contamination levels are still high, and large-scale production in bioreactors is not yet fully developed.…”

Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells

“…Additionally, most of the population has been exposed to natural adenovirus infection, therefore recombinant adenovirus vectors are likely to encounter neutralizing anti-adenovirus antibodies in patient plasma. 4,5 To avoid pre-existing humoral immunity in patients, several groups have investigated adenovirus serotypes from alternative species such as cows, [6][7][8][9][10][11][12][13][14] sheep, [15][16][17][18][19][20][21][22][23][24][25] chimpanzees, [26][27][28] dogs [29][30][31][32][33][34][35][36] and chickens. [37][38][39][40] Chicken embryo lethal orphan virus (CELO; fowl adenovirus type 1), characterized as an infectious agent in 1957, 41 is attractive as a gene delivery vector since it is simple and cheap to produce in bulk in chicken embryos and has a good safety profile being completely replication defective in human cells, even in the presence of wild-type human adenovirus.…”