Archive for February 8th, 2010

Re-engineering AAV

8 February, 2010

Adeno-associated virus (AAV) virion. Copyright Russell Kightley Media

Tweaking virus vectors used for gene therapy to change their receptor specificity is not necessarily new – but it has seldom been done (at least, to my mind) as elegantly as is reported in January’s Nature Biotechnology.  Asokan et al. report on

Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle
Nature Biotechnology 28, 79 – 82 (2010)
Published online: 27 December 2009 | doi:10.1038/nbt.1599

From the abstract:

We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism.

What impressed me most about the paper was the excellent modelling graphics: the authors were able to show, in simple 3-D atomic models, just how their mutations had changed the surface archotecture of the virus in question.  The whole-animal imaging was also very useful in showing very simply how effective their different constructs were.

(a) Three-dimensional structural model of the AAV2 capsid highlighting the 585–590 region containing basic residues implicated in heparan sulfate binding. Inset shows VP3 trimer, with residues 585-RGNRQA-590 located on the innermost surface loop highlighted in red. VP3 monomers are colored salmon, blue, and gray. Images were rendered using Pymol. (c) Representative live animal bioluminescent images of luciferase transgene expression profiles in BALB/c mice (n = 3) injected intravenously (tail vein) with AAV2, AAV8, AAV2i8 and structurally related AAV2i mutants (dose 1 × 1011 vg in 200 μl PBS) packaging the CBA (chicken beta actin)-Luc cassette. All AAV2i mutants show a systemic transduction profile similar to that of AAV8, with AAV2i8 showing enhanced transduction efficiency. Bioluminescence scale ranges from 0–3 × 106 relative light units (photons/sec/cm2). Residues within 585–590 region in each AAV2i mutant is indicated below corresponding mouse image data. (d) Comparison of AAV2, AAV2i8 and AAV8 capsid surface residues based on schematic “Roadmap” projections. A section of the asymmetric unit surface residues on the capsid crystal structures of AAV2 and AAV8, as well as a model of AAV2i8, are shown. Close-up views of the heparan sulfate binding region and residues 585–590 reveal a chimeric footprint on the AAV2i8 capsid surface. Red, acidic residues; blue, basic residues; yellow, polar residues; green, hydrophobic residues. Each residue is shown with a black boundary and labeled with VP1 numbering based on the AAV2 capsid protein sequence.

Adapted by permission from Macmillan Publishers Ltd: Nature Biotechnology 28, 79 – 82 copyright (2010)

Changing the tissue specificity of a well-characterised and often-used vector virus such as AAV in this way is an extremely useful thing to have done: it probably lowers the potential toxicity of the vector – by avoiding the liver – while preserving useful features such as the higher-level expression afforded by use of AAV2.

…and here they come…

8 February, 2010

…Big Pharma, to the biopharming revolution, that is.  Following on from earlier news about Protalix’s glucocerebrocidase successes, comes this latest snippet from Nature Biotechnology:

Pfizer stakes a claim in plant cell–made biopharmaceuticals

Mark Ratner

On December 1, Pfizer became the first big pharma to commit to take to market a late-stage biologic drug produced in plant cells. It acquired rights to taligurase alfa, a form of the enzyme glucocerebrosidase in development for the treatment of Gaucher’s disease, from Protalix Biotherapeutics in Carmiel, Israel. Protalix has completed phase 3 studies and has submitted a new drug application for the drug, also known as prGCD, eyeing US marketing approval in 2010. At the request of the US Food and Drug Administration (FDA) last year, the company has already begun supplying prGCD to patients in the US under an expanded access program and similarly to patients in the EU under a compassionate-use protocol [my emphasis – Ed].

Very interesting development, this: while Bayer led the way in acquiring plant expression vector maestros Icon Genetics a couple of years ago, there has not been much interest by Bigger Pharma in getting hold of the plant-expressed biologics startups – but expect things to change, starting now.

Ratner goes on, quoting Yuri Gleba of Icon / Bayer:

…Icon’s Gleba acknowledges that Protalix’s success in product development with prGCD shows they have a keen business sense. “If you are not strong on one side you have to compensate by being excellent on another, and by all accounts, they are,” he says. The deal with Pfizer and the approval of prGCD “should open the floodgates, in my opinion,” he says. “It is by far the most significant development in the plant-made pharmaceuticals arena right now.”

The floodgates are opening; Big Pharma is at the door – finally!