A functional mini-GDE transgene corrects impairment in models of glycogen storage disease type III.

Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). No curative treatment exists for GSDIII. The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus-derived (rAAV-derived) vector gene therapy strategy. Using information on GDE structure and molecular modeling, we generated multiple truncated GDEs. Among them, an N-terminal-truncated mutant, ΔNter2-GDE, had a similar efficacy in vivo compared with the full-size enzyme. A rAAV vector expressing ΔNter2-GDE allowed significant glycogen reduction in heart and muscle of Agl-/- mice 3 months after i.v. injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl-/- rat model. Finally, transduction with rAAV vectors encoding ΔNter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII. In conclusion, our results demonstrated the ability of a single rAAV vector expressing a functional mini-GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to patients with GSDIII.

Analysis of Adeno-Associated Virus Serotype 8 (AAV8)-antibody complexes using epitope mapping by molecular imprinting leads to the identification of Fab peptides that potentially evade AAV8 neutralisation.

Gene therapy is a promising approach for treating genetic disorders by delivering therapeutic genes to replace or correct malfunctioning genes. However, the introduced gene therapy vector can trigger an immune response, leading to reduced efficacy and potential harm to the patient. To improve the efficiency and safety of gene therapy, preventing the immune response to the vector is crucial. This can be achieved through the use of immunosuppressive drugs, vector engineering to evade the immune system, or delivery methods that bypass the immune system altogether. By reducing the immune response, gene therapy can deliver therapeutic genes more effectively and potentially cure genetic diseases. In this study, a novel molecular imprinting technique, combined with mass-spectrometry and bioinformatics, was used to identify four antigen-binding fragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodies capable of binding to AAV. The identified Fab peptides were shown to prevent AAV8's binding to antibodies, demonstrating their potential to improve gene therapy efficiency by preventing the immune response.

Capsid-specific removal of circulating antibodies to adeno-associated virus vectors.

Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.

Toward a Scalable Purification Protocol of GaLV-TR-Pseudotyped Lentiviral Vectors.

Lentiviral vectors (LV) that are used in research and development as well as in clinical trials are in majority vesicular stomatitis virus G glycoprotein (VSVg) pseudotyped. The predominance of this pseudotype choice for clinical gene therapy studies is largely due to a lack of purification schemes for pseudotypes other than VSVg. In this study, we report for the first time the development of a new downstream process protocol allowing high-yield production of stable and infectious gibbon ape leukemia virus (GaLV)-TR-LV particles. We identified critical conditions in tangential flow filtration (TFF) and chromatographic steps for preserving the infectivity/functionality of LV during purification. This was carried out by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and by defining the optimal order of these steps. A three-step process was developed for GaLV-TR-LV purification consisting of one TFF and two chromatographic steps (ion-exchange chromatography and size exclusion chromatography) permitting recoveries of >27% of infectious particles. With this process, purified GaLV-pseudotyped LV enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin-1 peptide, whereas in the same conditions nonpurified vector transduced only 9% of the cells (multiplicity of infection 20). Our protocol will allow for the first time the purification of GaLV-TR-LV that are biologically active, stable, and with sufficient recovery in the perspective of preclinical studies and clinical applications. Obviously, further optimizations are required to improve final vector yields.

Origins of truncated supplementary capsid proteins in rAAV8 vectors produced with the baculovirus system.

The ability to produce large quantities of recombinant Adeno-Associated Virus (rAAV) vectors is an important factor for the development of gene therapy-based medicine. The baculovirus/insect cell expression system is one of the major systems for large scale rAAV production. So far, most technological developments concerned the optimization of the AAV rep and cap genes in order to be expressed correctly in a heterologous system. However, the effect of the baculovirus infection on the production of rAAV has not been examined in detail. In this study we show that the baculoviral cathepsin (v-CATH) protease is active on several (but not all) rAAV serotypes, leading to a partial degradation of VP1/VP2 proteins. Subsequently, we identified the principal v-CATH cleavage site in the rAAV8 capsid proteins and demonstrated that the cleavage is highly specific. The proteolytic degradation of VP1/VP2 AAV capsid proteins reduces the infectivity of rAAV vectors but can be prevented by the use of a baculovirus vector with a deletion of the chiA/v-cath locus or by addition of the E64 protease inhibitor during production. Moreover, the codon optimization of AAV cap performed for several serotypes and originally aimed at the removal of potential alternative initiation codons, resulted in incorporation of additional forms of truncated VP1 into the rAAV capsids.

Incorporation of 2,3-diaminopropionic acid into linear cationic amphipathic peptides produces pH-sensitive vectors.

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pK(a) of the beta-NH(2) in 2,3-diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pK(a) of around 6.3 is stabilized by the increased electron-withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large-scale conformational changes are observed in Dap-rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene-delivery capability on designed cationic amphipathic peptides that incorporate Dap.

Design and evaluation of histidine-rich amphipathic peptides for siRNA delivery.

PURPOSE: Short linear peptides have a high potential for delivering various drugs with therapeutic potential, including nucleic acids. Recently, we have shown that the cationic amphipathic histidine-rich peptide LAH4 (KKALLALALHHLAHLALHLALALKKA) possesses high plasmid DNA delivery capacities. Since such peptides are thought to efficiently disrupt endosomal membranes, we have tested their ability to deliver small interfering RNA (siRNA) into mammalian cells. METHODS: Using a human cell line stably transfected with a luciferase-encoding expression vector, we have evaluated the ability of LAH4 and five derivatives thereof to deliver siRNAs and silence gene expression. RESULTS: The six peptides are all efficient siRNA delivery vehicles whose efficiency in mediating gene silencing in 911-Luc cells was greater than that of commercially available compounds including Lipofectamine, DOTAP and polyethylenimine. In addition, by using the proton pump inhibitor bafilomycin A1, we show that efficient siRNA delivery to the cytosol requires acidification of the endosomes. CONCLUSIONS: The LAH4 histidine-rich cationic amphipathic peptides represent an interesting and promising family of compounds for siRNA delivery.