Vector systems for prenatal gene therapy: principles of adeno-associated virus vector design and production.

Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeutic gene transfer in extensive pre-clinical data and, recently, in clinical trials. Careful vector design and choice from a range of natural or synthetic pseudotypes allow targeted, efficient, and sustained expression of therapeutic genes. The efficiency of gene delivery can be further enhanced through the use of drug pre-treatment or co-infection with a suitable helper virus. This chapter describes current best practice for AAV production, including complete methods for: (1) efficient generation of vector without the use of helper viruses, simplifying the transition to GMP-grade production for clinical applications; (2) efficient and easily scalable purification of the virus by affinity chromatography, allowing rapid production of highly concentrated, high titre stocks; (3) reliable quantification and assaying of viral stocks, along with short- and long-term storage considerations.

AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage.

We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.

A multiscale mathematical model for oncolytic virotherapy.

One of the most promising strategies to treat cancer is attacking it with viruses. Oncolytic viruses can kill tumor cells specifically or induce anticancer immune response. A multiscale model for virotherapy of cancer is investigated through simulations. It was found that, for intratumoral virus administration, a solid tumor can be completely eradicated or keep growing after a transient remission. Furthermore, the model reveals undamped oscillatory dynamics of tumor cells and virus populations, which demands new in vivo and in vitro quantitative experiments aiming to detect this oscillatory response. The conditions for which each one of the different tumor responses dominates, as well as the occurrence probabilities for the other nondominant therapeutic outcomes, were determined. From a clinical point of view, our findings indicate that a successful, single agent virotherapy requires a strong inhibition of the host immune response and the use of potent virus species with a high intratumoral mobility. Moreover, due to the discrete and stochastic nature of cells and their responses, an optimal range for viral cytotoxicity is predicted because the virotherapy fails if the oncolytic virus demands either a too short or a very large time to kill the tumor cell. This result suggests that the search for viruses able to destroy tumor cells very fast does not necessarily lead to a more effective control of tumor growth.