Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

Bioprocess development for canine adenovirus type 2 vectors.

Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors produced in MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ∼10(9) infectious particles (IP) ml(-1) and 2 × 10(3) IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.

Human innate lymphoid cell activation by adenoviruses is modified by host defense proteins and neutralizing antibodies.

Innate lymphoid cells (ILCs), the complements of diverse CD4 T helper cells, help maintain tissue homeostasis by providing a link between innate and adaptive immune responses. While pioneering studies over the last decade have advanced our understanding how ILCs influence adaptive immune responses to pathogens, far less is known about whether the adaptive immune response feeds back into an ILC response. In this study, we isolated ILCs from blood of healthy donors, fine-tuned culture conditions, and then directly challenged them with human adenoviruses (HAdVs), with HAdVs and host defense proteins (HDPs) or neutralizing antibodies (NAbs), to mimic interactions in a host with pre-existing immunity. Additionally, we developed an ex vivo approach to identify how bystander ILCs respond to the uptake of HAdVs ± neutralizing antibodies by monocyte-derived dendritic cells. We show that ILCs take up HAdVs, which induces phenotypic maturation and cytokine secretion. Moreover, NAbs and HDPs complexes modified the cytokine profile generated by ILCs, consistent with a feedback loop for host antiviral responses and potential to impact adenovirus-based vaccine efficacy.

Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n.

The sequence of a Pst I restriction fragment was determined that demonstrate instability in fragile X syndrome pedigrees. The region of instability was localized to a trinucleotide repeat p(CCG)n. The sequence flanking this repeat were identical in normal and affected individuals. The breakpoints in two somatic cell hybrids constructed to break at the fragile site also mapped to this repeat sequence. The repeat exhibits instability both when cloned in a nonhomologous host and after amplification by the polymerase chain reaction. These results suggest variation in the trinucleotide repeat copy number as the molecular basis for the instability and possibly the fragile site. This would account for the observed properties of this region in vivo and in vitro.

The conundrum between immunological memory to adenovirus and their use as vectors in clinical gene therapy.

In the context of clinical gene transfer using viral vectors, the risk of memory antivector immunity is often poorly appreciated. The immunological past of the patient, the site of injection, and the vector dose will play intertwined and decisive roles in the safety and efficacy of treatment. To circumvent the drawbacks due to the ubiquitous human adenovirus (HAd) memory immunity, we believe that vectors derived from canine adenovirus type 2 (CAV-2) will be more clinically useful than those derived from HAds based, in part, on the potential lack of immunological memory. CAV-2 is not a human pathogen in spite of the approx 100,000 yr of cohabitation of humans with dogs. During the last 8 yr, we found that CAV-2 vectors preferentially transduced neurons in the central nervous system (CNS) of several species, and had a surprisingly efficient level of axoplasmic transport. CAV-2 vectors also lead to greater than 1 yr transgene expression in the immunocompetent rat CNS-without immunosuppression. However, more immediate harm can be caused to a patient via an acute and/or chronic vector-induced cellular infiltration in the CNS than by the normal progression of most neurodegenerative disorders. In this context, we continue to assess the clinical potential of CAV-2. This mini-review addresses our analysis of the interaction of CAV-2 vectors with human memory immunity and monocyte-derived dendritic cells.

Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo.

In the central nervous system (CNS), there are innate obstacles to the modification of neurons: their relative low abundance versus glia and oligodendrocytes, the inaccessibility of certain target populations, and the volume one can inject safely. Our aim in this study was to characterize the in vivo efficacy of a novel viral vector derived from a canine adenovirus (CAV-2). Here we show that CAV-2 preferentially transduced i) rat olfactory sensory neurons; ii) rodent CNS neurons in vitro and in vivo; and, more clinically relevant, iii) neurons in organotypic slices of human cortical brain. CAV-2 also showed a high disposition for retrograde axonal transport in vivo. We examined the molecular basis of neuronal targeting by CAV-2 and suggest that due to CAR (coxsackie adenovirus receptor) expression on neuronal cells-and not oligodendrocytes, glia, myofibers, and nasal epithelial cells-CAV-2 vectors transduced neurons preferentially in these diverse tissues.

Trafficking and propagation of canine adenovirus vectors lacking a known integrin-interacting motif.

Canine adenovirus type 2 (CAV-2) vectors may be attractive tools for gene transfer thanks to the lack of pre-existing immunity in humans, and because of the preferential transduction of neurons when the vector is injected into the brain and some innervated tissues. The coxsackievirus-adenovirus receptor (CAR) appears to play a major role during infection of most human serotypes, whereas the alpha(v)beta(3/5) integrins have been reported to play a significant auxiliary role. We showed that CAV-2 also attaches to and uses CAR to enter cells, but CAV-2 transduction could be notably different from that of the prototype human adenovirus serotype 5 (Ad5). Initially, the CAV-2 capsid appears to be 10-fold less negatively charged than Ad5. Second, the CAV-2 penton, hexon, and fiber proteins do not contain a known integrin-interacting motif. Because of its potential interest in the clinic, we analyzed the different steps of cellular trafficking and the propagation kinetics of CAV-2 vectors. We found that Ad5 and CAV-2 vectors have comparable kinetics of binding (10 min), internalization (10 min), endosomal escape (17 min), attachment to the nuclear membrane (35 min), and formation (18 hr) and release (34 hr) of functional virions. Surprisingly, the RGD(-) CAV-2 capsid also induced the reorganization of actin filaments in HeLa cells. Actin reorganization is thought to be dependent on alpha(v)beta(3/5) integrin stimulation.

A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.

Analysis of the promoter for the gene encoding the testis-specific histone H1t in a somatic cell line: evidence for cell-cycle regulation and modulation by distant upstream sequences.

Gene H1t encodes a testis-specific variant of the H1 histone family expressed in pachytene spermatocytes during the meiotic phase of spermatogenesis. Fusions between various upstream fragments of the H1t gene and the chloramphenicol acetyltransferase-encoding reporter gene were analyzed in mouse L cells by both transient and permanent transfection. Expression of the minimal promoter [174 nucleotides (nt) upstream from the transcription start point] was enhanced by sequences extending to nt -693, but was reduced in constructs with kb of upstream sequence. Using synchronized cells, expression was at least twofold higher in growing than in inhibited cells. Thus, the H1t promoter is modulated both positively and negatively by distant upstream sequences, and it displays some of the S-phase-dependent character of a replication-dependent histone.

Prevention of posterior capsule opacification by the induction of therapeutic apoptosis of residual lens cells.

Posterior capsule opacification (PCO) is a common complication of cataract surgery. Using adenovirus(Ad)-mediated gene transfer, we overexpressed the proapoptotic molecules p53, procaspase 3, Bax, and TRAIL to induce therapeutic programmed cell death of residual lens cells to prevent PCO. Overexpressed TRAIL did not induce apoptosis in cultured rabbit lens cells or in human lens cells. Overexpressed p53 induced apoptosis of lens cells in vitro and ex vivo, but was unable to prevent PCO in vivo. Overexpressed procaspase 3 was associated with engagement of many components of the apoptotic pathway, including cleavage of intracellular caspase targets such as PARP and inter-nucleosome DNA fragmentation. Even when only slightly overexpressed, Bax caused apoptosis of transduced rabbit and human lens cells by engaging the mitochondrial pathway, including catalytic activation of the caspases. A single in vivo injection of Ad vectors expressing either Bax or procaspase 3 into the capsular bag at the end of phacoemulsification prevented PCO in rabbits. These experiments show that Ad-mediated Bax or procaspase 3 overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo in residual lens cells and preventing PCO in a rabbit model of PCO. Manipulation of proapoptotic molecule expression could be a novel gene therapy approach for prevention of PCO.

Lens cell targetting for gene therapy of prevention of posterior capsule opacification.

Posterior capsule opacification is the main complication of cataract surgery. Using adenovirus-mediated gene transfer, we recently reported that it was feasible to prevent PCO by overexpressing pro-apoptotic molecules such as pro-caspase 3 or Bax in the residual lens epithelial cells post-cataract surgery. However, this approach is feasible only if gene transfer can be restricted to the residual cells responsible for PCO. Initially, we tested an adenovirus (human serotype 5, HAd5), a lentivirus (HIV) and an oncoretrovirus (MLV) vector for the their in vivo transduction efficiency of rabbit lens cells. We found that HAd5 vectors were the most efficient (>90% of the cells could be transduced). Six potential lens-specific promoters were then cloned into HAd5 vectors and assayed for their ability to target expression to a specific population of cells, using in vitro, ex vivo and in vivo rabbit tissues and human lens capsular bags. We found that the LEP503, MIP and Filensin promoters induced strong lens-specific expression of a reporter gene, in human lens cells. Following this ex vivo assay, we showed in a rabbit PCO model that gene transfer could be spatially restricted to the capsular bag by confining the vector with Matrigel. Our combined approach using a lens-specific promoter and a biocompatible gel should render feasible a novel therapeutic strategy for PCO that targets the remaining lens cells.

Localization of mRNA for testis-specific histone H1t by in situ hybridization.

H1t is a testis-specific H1 histone variant that appears in germ cells during the meiotic prophase of mammalian spermatogenesis. Using a tritiated antisense RNA probe, H1t mRNA was identified by in situ hybridization in the mid and late pachytene spermatocytes found in seminiferous tubules of approximately stages VII to XIII.

Adenovirus and adeno-associated virus mediated gene transfer.

In this review we describe current strategies for adenoviral mediated gene transfer (AMGT) and adeno-associated viral mediated gene transfer (AAVMGT). We consider the structure and molecular biology of adenoviruses and adeno-associated viruses and detail the current advantages and disadvantages of AMGT and AAVMGT. Potential solutions to some of the specific drawbacks to AMGT, including the development of new vectors, addition of gp19k, organoides, and the use of non-human adenoviral vectors, are discussed.

Canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer.

Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.

Characterization of cis-acting sequences involved in canine adenovirus packaging.

The cis-acting packaging domain in adenovirus serotype 5 (Ad5) is a series of redundant, albeit not functionally equivalent, "A-repeats" made up of the consensus sequence 5'-TTTGN(8)CG-3'. A-repeats may bind trans-acting factors that direct packaging of the adenovirus genome into the preformed capsid. To try to understand this basic mechanism, we examined the packaging domain from a nonhuman adenovirus. We delimited the canine adenovirus type 2 (CAV-2) packaging domain to within 156 bp via a conditional mutation based on the Cre/loxP excision. Using an insertion, deletion, and substitution strategy, we generated packaging-defective CAV-2 vectors. Our results demonstrate that, like Ad5, CAV-2 cis-acting packaging sequences are located near the left inverted terminal repeat and are redundant, but not functionally equivalent. However, the bipartite motif found in Ad5 is present only once in CAV-2 and deletion of it caused only a minor variation in the packaging efficiency. We have identified at least four functional cis-acting packaging sequences in CAV-2. The CAV-2 vectors that we generated were not replication-defective in an E1-transcomplementing cell line and as heat stable as the parental vectors that did not contain mutations.

Adenovirus vector-transduced hepatocytes implanted via a preformed collagen/PTFE support persist for at least 4 weeks in vivo.

Liver-directed gene therapy has the potential to provide an effective adjutant to conventional treatments for liver diseases. In vivo gene transfer is promising but still effectively out of reach with conventional gene delivery techniques. Ex vivo gene therapy using hepatocyte transplantation, however, has been encouraging. We describe and approach toward treatment of liver-related diseases by combining several of the advantageous properties of current liver-directed therapies. Primary hepatocytes were isolated, transduced with an adenovirus vector encoding a reporter gene, embedded in a collagen/polytetrafluoroethylene (PTFE) lattice, and implanted in mice. Recovered 'hepatocyte-organoids' were assayed for the presence and viability of the implanted hepatocytes, duration of transgene expression and presence of the adenovirus vector. In an initial attempt, we demonstrate that genetically modified hepatocytes can survive and express a transgene for at least 4 weeks in vivo when embedded in a collagen/PTFE support and implanted in the intraperitoneal cavity. This approach takes advantage of hepatocyte-specific functions in order to treat diseases where a fraction of the normal enzymatic activity is sufficient to alleviate a disease phenotype.

Organoids direct systemic expression of erythropoietin in mice.

Organoids are adenoviral vector transduced cells embedded ex vivo in a collagen-polytetrafluoroethylene lattice that is saturated with angiogenic factors. Organoids provide an alternative method of cell mediated gene transfer following implantation in the donor/recipient. The feasibility of adenovirally mediated delivery via organoids using the erythropoietin (Epo) cDNA was tested. Fibroblasts were transduced by two recombinant adenoviral vectors encoding the Macaca cynomolgus Epo cDNA, driven by a viral (RSV LTR) or a murine housekeeping gene promoter (PGK-1). A functional in vivo assay was used to monitor Epo production via the rise in hematocrit(s) (hct). The hct remained elevated for as long as 6 weeks after implantation. Subcutaneous implants gave consistently higher hct than intraperitoneal implants, while organoids made with a greater number of cells, or an equal number of cells transduced at higher multiplicities of infection (MOI) also produced a larger increase in hct. AdPGKEpo-organoids produced a greater increase in hct than AdRSVEpo-organoids under comparable conditions, but the duration of expression was similar. A 10- to 50-fold lower input of AdRSVEpo using organoids versus direct intravenous injections resulted in an equal to, or greater than hct response in mice. Explanted organoids caused a rapid decrease in the hct of mice. Organoid supernatant had little or no detectable free viral particles making this method safe from unwanted recombinant adenovirus dissemination.

Expression of the rat testis-specific histone H1t gene in transgenic mice. One kilobase of 5'-flanking sequence mediates correct expression of a lacZ fusion gene.

H1t is synthesized in mid to late pachytene spermatocytes of the male germ line and is the only tissue-specific member of the mammalian H1 histone family. As a step toward identifying DNA sequences that confer its tissue-specific expression, we have produced transgenic mice containing the intact rat H1t gene as well as a H1t-lacZ fusion gene. Transgenic mice carrying a 6.8-kilobase fragment of rat genomic DNA encompassing the H1t gene expressed rat H1t at high levels in the testis and in no other organ examined. H1t fragments truncated to within 141 base pairs (bp) of the gene in the 5' direction or within 837 bp in the 3' direction retained testis specificity. Expression of rat H1t protein was also evident in the testes of the transgenic mice, and in some lines the level of rat H1t exceeded that of the mouse protein. The stage of spermatogenesis of transgene expression was assessed by following appearance of transgenic mRNA in developing mice and by immunohistochemistry using an antiserum to rat H1t. In lines from three different constructs, expression was restricted to germinal cells, although in two strongly expressing lines the transgenes were expressed somewhat prematurely in preleptotene spermatocytes. An H1t(-948/+71)-lacZ fusion was also expressed specifically in the spermatocytes and round spermatids of a transgenic line, confirming that sequences sufficient for correct tissue and developmental expression lie within this 1,019-bp segment of the gene.

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway.

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.