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Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors¹

CLAIRE SOUDAIS^*, NADIA SKANDER and ERIC J. KREMER^,2

^* INSERM Unit 550, Faculté de Médecine Necker, Paris 75015, France; and
Institut de Génétique Moléculaire de Montpellier, CNRS 5535, IFR 122 Montpellier 34293, France

² Correspondence: IGMM CNRS UMR 5535, 1919 Route de Mende, Montpellier 34293, France. E-mail: kremer{at}igm.cnrs-mop.fr

SPECIFIC AIMS

Our aims were i) to generate high-titer, helper-dependent CAV-2 vectors with a minimal level of contaminating helper vector using ii) a novel strategy to iii) test the in vivo efficacy of gene transfer in the CNS of rats, in particular the duration of expression and iv) the ability to safely increase the level of gene transfer

PRINCIPAL FINDINGS

1. Generation of plasmids containing gutless vectors
Subcloning into a 27 kb plasmid is challenging due to the paucity of convenient restriction enzyme sites. In addition, when generating helper-dependent constructs it is necessary to keep a consistent size for packaging efficiency and to better separate the helper-dependent and helper vectors during subsequent CsCl density gradients. We created a simple and flexible strategy that allows generation of helper-dependent constructs that are of optimal size. Depending on the size of the transgene expression cassette, one of a series of "pGut" is used for the initial subcloning step. This plasmid (e.g., pGut-1-containing GFP, pGutGFP) was linearized and used in homologous recombination in Escherichia coli with a plasmid containing the CAV-2 inverted terminal repeats, packaging domain and stuffer sequence (e.g., pEJK25). We simultaneously inserted the expression cassette and deleted the appropriate size fragment from the stuffer sequence to generate pSpike, a gutless construct containing a GFP expression cassette. This strategy has a user-friendly multiple cloning site, can be modified to accommodate any stuffer sequence, and keeps the gutless vector 27 kb (an optimal size for separation by CsCl density gradients).

2. Generation and characterization of improved helper vectors
To generate helper-dependent CAV-2 vectors, DKZeo (E1-transcomplementing) or DKCre cells (Cre-expressing/E1-transcomplementing) were transfected with linear pSpike and then infected by a helper vector. We generated and tested 3 helper vectors: JBlox, JB5, and JB19, which are 34 kb and contain floxed packing domains and a RSV-lacZ expression cassette. JBlox contains a wild-type packaging domain; JB5 and JB19 contain packaging mutants that are deleted in 4 or 6 of the 10 consensus packaging motifs. Spike is propagated by coreplication with the helper vector and preferentially packaged via the removal of the helper vector’s floxed packaging domain. Initial preparations of Spike using JBlox gave relatively low titers (5x10⁹ infectious units (i.u.)/mL) after amplification in DKCre cells and a high level (up to 25%) of contaminating JBlox. We then modified the strategy: we transfected and amplified (4 times) Spike in DKZeo cells, included a sorting of GFP⁺ cells by flow cytometry at each amplification, used JB5 or JB19 as the helper vector, and finally removed the helper vector with a final amplification in DKCre cells. The helper-dependent vectors were concentrated and purified on CsCl density gradients.

Using this approach a pronounced quantitative and qualitative effect of the advantages of JB5 and JB19 was seen: we were able to amplify Spike in fewer steps (6 vs. 10) than when using JBlox. In addition, JB5 and JB19 increased the production of Spike by three to fourfold (2 x10¹¹ particles/mL with 2.5x10¹⁰ i.u./mL of Spike and decreased the contamination of helper vector to 1 ß-galactosidase i.u.). We estimate that 1 i.u. of JB5 corresponds to 0.1 to 0.01% Ad5 helper plaque-forming units and is approximately the level found in human Ad helper-dependent vectors.

Finally, although wild-type CAV-2 cannot propagate in human cells, replication competent Ads (RCA) containing a potentially oncogenic E1 region are an unwelcome contaminant. We were unable to detect RCAs in 10¹¹ particles using a vector essentially identical to JBlox after repeated serial amplifications. Thesedata suggested that our vector/cell line combination is unable to generate RCAs.

3. Duration, distribution, and efficacy of transgene expression in vivo
Adult Sprague Dawley rats were injected with Spike in the striatum and killed at wk 2, 6, 26, or 52. At 2 wk postinjection, we found a level of GFP expression similar to CAVGFP (an E1-deleted CAV-2 vector expressing GFP) (Fig. 1A-D ). In contrast to CAVGFP, at 6 wk and 6 months postinjection of Spike (Fig. 1E-F ) we still found a high level of expression that was 60 to 70% the level seen at wk 2.

View larger version (62K): [in this window] [in a new window]   Figure 1. In vivo analysis of Spike after stereotactic injection in the rat brain. A–D) At wk 2, serial sections of the SNpc of a Sprague Dawley rat after injection in the striatum with 107 i.u. of Spike. GFP+ cells could be found from the beginning to the end of the SNpc, E) at 6 wk, F) 6 months, and G) 1 year. H) GFP+ cells in the striatum (original magnification 45x) at 1 year. We estimated that there were at least 600 transduced cells at wk 2 in the SNpc. A second cohort of rats were injected at 3 distinct sites in the right and a single site in the left striatum with 107 i.u./site and killed 4 wk postinjection in order to try to increase the number of modified cells: I) a single injection, and J) triple injection in the same rat. We estimated that there were >3000 transduced cells in the right hemisphere. An eightfold higher magnification K) of a section of the SNpc from the right hemisphere is also shown. L) Schematic shows the site of injections in the striatum*(blue), the location of the SNpc* (green), and the basal nuclei of Meynert* (red).

We then asked three complementary questions: 1) is it possible to inject at multiple sites without significant toxicity; 2) do multiple injections lead to higher levels of transduction of the SNpc; and 3) because the striatum is not homogeneous, could we target other basal nuclei? We stereotactically injected 2 cohorts of rats: the first with a single injection in the left striatum, and the second at 3 sites in the right and 1 sit in the left hemisphere. By injecting at 3 separate coordinates in the striatum we were able to transduce 5-fold more dopaminergic (DA) neurons in the SNpc than with a single injection (Fig. 1I-K ). We found that >3000 of the rat’s 6000 SNpc DA neurons were transduced. In addition to the transduction of the SNpc, we also found a high level of transduction of the cholinergic neurons in the basal nuclei of Meynert, via the more caudal injection site in the striatum. To our knowledge, this is the first description of retrograde transport of a vector from the striatum to this basal nucleus.

One year after striatal injections, we found stable, high-level expression in striatal neurons (Fig. 1H ), in the dopaminergic neurons of the SNpc (Fig. 1G ), and in the cholinergic neurons in the basal nuclei of Meynert.

CONCLUSIONS AND SIGNIFICANCE

Effective treatment of complex neurological disorders is critical to human integrity. Numerous causes, variable pathophysiologies, the blood-brain barrier, and the complex and fragile neural circuitry create a formidable challenge for the treatment of neurodegenerative diseases. Although there are intracellular strategies to address neurodegeneration, one fundamental criterion for the long-term survival of neurons may be their genetic modification. If one wanted to genetically modify neurons, for basic research or therapeutic reasons, the vector should preferentially modify these highly differentiated cells, which make up 10% of the primate brain. To exploit the neural tropism of CAV-2 vectors, we generated vectors that could generate long-term, stable in vivo expression (Fig. 2 ). In a previous study, CAVGFP generated strong expression at wk 2, but progressively diminished until little transgene expression could be detected at 3 months.

View larger version (38K): [in this window] [in a new window]   Figure 2. Schematic diagram.

Due to the intrinsic properties of 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. More optimistically, we suggest that helper-dependent CAV-2 vectors are clinically relevant vectors for the therapy of some neurodegenerative disorders. The advent of helper-dependent vectors has eliminated the majority of the problems associated with the induced CD8⁺-mediated destruction of transduced cells in naive immunocompetent animals, and long-term, stable transgene expression is possible. Nonetheless, the preexisting humoral and long-lived cellular immunity found in the majority of potential patients are likely to limit the clinical advantages of vectors derived from human viruses (like Ad5). The novel aspect of our work is the creation and characterization of vectors needed for the safe, efficient, and long-term genetic modification of terminally differentiated neurons. Via selective stereotactic injection, one may be able to target distal populations of cell, by injecting regions that have axonal projections. This advantage should not be underestimated. In most cases, the complex and fragile neural circuitry of the CNS should not be damaged during the injection. This is primarily why high titer stocks are indispensable: small volumes and slow delivery times are needed in order to reduce compression damage in the CNS. An obvious target for CAV-2 vectors is to help understand the pathophysiology of Parkinson’s disease. Selective modification of the SNpc in a single hemisphere, and testing the long-term or temporal effects of environmental factors on modified vs. unmodified neurons, could be accomplished. With the large cloning capacity, several genes could be assayed simultaneously.

Because of the high level of retrograde transport in many types of neurons of afferent structures, another excellent target is neurodegenerative damage caused by certain lysosomal storage disorders (LSD) where global enzyme expression may be needed. Lysosomal enzymes can be involved in the intracellular degradation of macromolecules to low molecular weight compounds. Deficiencies of these enzymes result in the accumulation of undegraded macromolecules within the lysosomes, leading to the subsequent pathological features of the disease. Usually there is a specific enzyme deficiency, but there may be a failure of delivery of enzymes to the lysosomes, defective transport of a small molecule out of lysosomes, or deficiency of a small molecular weight protein that participates in degradation of sphingolipids. The pathophysiology of some LSDs, a collection of 40 orphan diseases where the neurological symptoms can be dominant, makes them excellent candidates for gene therapy because there is no available treatment of the neurodegeneration. Davidson et al. elegantly demonstrated that ß-glucuronidase activity could be restored in ß-glucuronidase-deficient mice injected with adeno-associated virus or lentivirus vectors and a phenotypic correction was possible. As encouraging as the results are, the challenges of global correction in the mouse brain, which is smaller than the end of one’s thumb, does not adequately address the difficulties of treating a human brain. Current vectors (e.g., lentiviruses, oncoviruses, parvoviruses, herpes, and adenoviruses) all have their individual conveniences and disadvantages. Notably, lentivirus (HIV, SIV and FIV) and parvovirus (AAV) vectors do not traffic via axonal transport and modify cells only at the site of injection. The intrinsic properties of CAV-2 vectors, in synergy with the distribution of lysosomal enzymes via axonal transport, would make a novel strategy for LSD therapy.

We are continuously moving toward vectors that fill requirements based on the pathophysiology of specific diseases. Nonhuman viral vectors are being evaluated with increasing interest and surprises. It was clearly a serendipitous finding that CAV-2, which naturally infects the upper respiratory track of young dogs, had potential clinical application in neurodegenerative diseases. The work described here will allow us to address questions concerning virology, neurobiology, immunology, cell biology, as well as clinical medicine.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0438fje

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