Mention the word ‘archaeology’ and ancient civilisations, excavations and Indiana Jones (or Lara Croft) usually spring to mind. Molecular archaeology, however, describes the study of matter that is invisible to the naked eye; instead of trowels and brushes, molecular archaeologists use sophisticated laboratory tools to understand how the building blocks of life have evolved over time. In a recent study published in Cell Reports (Zinn et al., 2015) senior author Dr. Luk Vandenberghe uses a molecular archaeology approach to resurrect an extinct adeno-associated virus, with a view to improving existing gene therapies.
The holy grail of gene therapy is to find a vector, or carrier, that can efficiently shuttle therapeutic genes into diseased tissues, evading detection and destruction by the human immune system. The discovery and vectorisation of adeno-associated viruses (or AAVs) could be viewed as the first steps towards this holy grail: efficient, non-pathogenic and showing little toxicity, AAVs quickly emerged as key players in gene therapy clinical trials.
Trials using AAVs to treat diseases such as haemophilia B, heart failure and muscular dystrophy are currently underway, providing hope to thousands of patients suffering from these incurable and frequently debilitating conditions. A major obstacle to the use of AAV in the clinic is the existence of anti-AAV immunity, which arises when people have previously been exposed to these naturally-occurring viruses. This significantly reduces the efficacy of AAV gene therapies, rendering such patients ineligible for treatment. Efforts to evade pre-existing patient immunity by modifying individual components of the AAV capsid (or shell) have been hampered by the intricate structure of these viruses: like a 3-D jigsaw, the integrity of the icosahedral AAV capsid is dependent on each component part fitting together perfectly.
That brings us back to Dr. Vandenberghe’s study. Rather than performing forward genetic studies to create novel AAVs, Dr. Vandenberghe used a form of molecular time-travel known as maximum likelihood-ancestral sequence reconstruction (or ML-ASR [Finnigan et al., 2014]) to predict the protein sequences of long-extinct AAVs. He reasoned that by looking to the past for clues on how AAV capsid proteins fit together, he would find AAV ancestors that could evade present-day patient immunity, while retaining structural and functional integrity.
An AAV evolutionary tree was generated by analysing the Cap protein sequences from 75 different AAV serotypes, including many of the AAVs currently used in gene therapy trials. By tracing back through the AAV lineage, Dr. Vandenberghe’s group was able to identify a single common ancestor, Anc80, from which most contemporary AAVs have evolved. As there was some ambiguity over the identity of certain amino acids in the protein sequence, a library containing the 2048 possible permutations was constructed. Of these, Anc80L65 was selected for further evaluation, based on its ability to assemble into stable, intact AAV particles that efficiently infected cells in culture.
Crucially, when the group tested the resurrected AAV in vivo, they observed high levels of transgene expression in the mouse retina, skeletal muscle and liver – showing that Anc80L65 could act as a highly-efficient vector for gene delivery to diseased tissues. Importantly, no evidence of Anc80L65 toxicity was found, and when Anc80L65 was administered to mice that had previously been immunised against present-day AAV serotypes, only partial Anc80L65 neutralisation was observed. Conversely, AAV8 (a commonly-used gene therapy vector currently in clinical trials for haemophilia B) was almost completely neutralised in pre-immunised mice, confirming that Anc80L65 is an immunologically distinct virus which may be able to circumvent the largest remaining obstacle to effective AAV gene therapy.
Through this ingenious act of molecular time-travel, it appears that Dr. Vandenberghe has uncovered a virus with great potential for future gene therapy applications.
Finnigan, G.C., Hanson-Smith, V. et al., (2014). Evolution of increased complexity in a molecular machine. Nature. 481, 360-365.
Zinn, E., Pacouret, S. et al., (2015). In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector. Cell Reports. 12, 1056-1068.