Molecular breeding of viruses

Abstract

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection

“…For construction of random chimeric AAV capsid gene libraries, AAV serotypes 1, 2, 3B, 4, 6, 7, 8, and 9 were used as PCR templates. The capsid genes were amplified by primers CAP-5Ј (5Ј-CCC-AAGCTTCGATCAACTACGCAGACAGGTACCAA-3Ј) and CAP-3Ј (5Ј-ATAAGAAT-GCGGCCGC-AGAGACCAAAGTTCAACTGAAACGA-3Ј) and mixed in equal ratio for DNA shuffling (18). In brief, 4 g of the DNA templates were treated by 0.04 U of DNase I at 15°C briefly.…”

“…The structural information on the capsid forms the basis for genetic engineering of novel AAV vectors. DNA shuffling has recently been used to modify viral vectors (18,19) by introducing enormous permutations of genetic variations via in vitro recombination. The shuffled AAV mutant libraries were used for selection of desirable features such as resistance to antibody neutralization (19,20) and improved tropism to cancer cells (21).…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…Such family shuffling can engender variants with novel properties not possessed by any of the parents. Soong et al (2000) first generated a random chimeric retroviral envelope library from six distinct MLV parent sequences and selected it for a novel function, the ability to support murine leukemia virus (MLV) infection of Chinese hamster ovary (CHOK1) cells, normally non-permissive to MLV infection. Even though detailed mechanisms involved in the new virus tropism, such as the cell surface receptor utilized by the new variants, were not explored, this work showed a great potential of gene shuffling to generate viral envelope proteins with novel and desirable properties.…”

Progrès scientifiques et nouvelles armes biologiques

“…Découverte en 1994 par Willhem Stemmer du Affymax research institute à Palo Alto (Californie), une méthode appelée « évolution dirigée par élevage moléculaire » [19,20] (DNA shuffling) permet de fabriquer, en un court laps de temps, des germes aux propriétés nouvelles. En découpant plusieurs gènes très proches en tout petits fragments, on peut reconstituer au hasard des gènes entièrement nouveaux, que l'on peut ensuite sélectionner in vitro : une floraison de publications, parues dans la littérature scientifique depuis quelques années, sous l'égide de start-up américaines de biotechnologie, expliquent avec force détails comment créer rapidement des gènes, des bactéries, des virus ou des toxines aux propriétés « améliorées », voire entièrement nouvelles : germes (bactéries ou virus) très résistants à des conditions très hostiles (acidité…) ou aux substances anti-infectieuses disponibles (une nouvelle souche de colibacille 32 000 fois plus résistante aux pénicillines [21] a ainsi facilement été isolée, alors que les méthodes traditionnelles par mutations in vitro ne permettent qu'une augmentation d'un facteur 16) ; virus aux propriétés nouvelles [22] (l'affinité tissulaire de rétrovirus humain et murin a été modifiée, leur permettant de se répliquer dans des cellules non permissives au virus sauvage [23,24]). Ces dernières données montrent qu'il est désormais possible d'influencer le tropisme des virus, et l'on pressent les conséquen-ces catastrophiques de la transformation d'un virus à transmission sexuelle en un virus transmis par voie aérienne.…”