Tuesday, November 20, 2012

Demystifying ddRNAi Trigger Design

A somewhat vexing problem with DNA-directed RNAi (ddRNAi) has been the heterogeneous mixture of small RNAs generated from a typical expression vector.  Not only will this compromise knockdown efficacy through competition for RISC loading, it also poses a safety risk by increasing the number of off-target genes.  For a long time, heterogeneous ddRNAi trigger processing had been accepted as a way of life- as long as the efficacy was right you would not waste too many thoughts on those multiple bands on your Northern blot.  

Choosing the double-strand length of a ddRNAi trigger has also been more art than science.  Different sizes, usually between 19 and 29bp facilitate potent gene knockdown, but there have been few studies looking at the consequence of size on the uniformity of hairpin processing.   

Next-generation sequencing technology is changing these attitudes.  In a paper that appeared last week in CELL, Gu et al. from the Kay lab in Stanford showed that next-gen sequencing is a powerful tool to detect which small RNA species are generated from a given ddRNAi template plus their relative quantities.  Moreover, through an iterative process of structural change and sequencing, a key structural feature causing the Dicer enzyme make just one predictable cut could be identified.  Without going into much detail, a simple 21 base-pair hairpin yields the purest results.

Commercial ddRNAi trigger landscape getting rusty  

While the clinical validation of SNALP delivery technology has allowed synthetic RNAi trigger-based Tekmira and Alnylam to turn around their fortunes and should also increase general interest in the technology, there seem to be no commercial players left in the ddRNAi Therapeutics arena able to champion and refine the platform. 

Benitec is pre-occupied with trying to monetize IP that is rapidly losing in value due to its age and as a result failing to become a real biotechnology company with a lab.  The two viral delivery companies that once had some ddRNAi ambitions, AMT (AAV) and Oxford Biomedica (lentivirus), either went out of business and/or lack the requisite RNAi molecular biology expertise.  And the other groups developing ddRNAi Therapeutics, including Calimmune and Genables are de facto one-product, disease-focused companies.  This is unfortunate as there should be room for at least one or two ddRNAi Therapeutics platform companies.

Such a company would have expanded on the basic dsRNA concept and would thus have kept its IP estate fresh, eventually forcing others to take a license as simply running out the patent clock would not have been an option.  Such IP would include discoveries like the one made by Gu and colleagues.  On a more positive note, it is possible that the current revival of gene therapy after a decade of neglect and scorn will eventually carry ddRNAi Therapeutics along with it.


Anonymous said...

Benitec recently acquired the Hep C program developed by Pfizer which is ready to go to clinical trials in the new year. the small cell lung cancer program demonstrated good delivery with a non viral vector and Gradalis continues with its ddRNA clinical programs so not all is amiss.

Anonymous said...

Benitec had the lab set up and ready to go in Cal, when Nucleonics somehow got USPTO to do a 6 year re-exam going on Benitec's IP.

Lab had to be shuttered before they had unpacked the boxes. And it was a big lab.

Benitec are securing IP in the new inhouse and out licenced programmes.

To open a lab now will put too much cash burn onto the books. The lab will come once they've re-established their cash foundation.

Obvious as much as it's prudent really.

Anonymous said...

If the HepC program that Benitec acquired from Pfizer was actually working, do you think Pfizer would have let it go??

Anonymous said...

Dirk, thanks for your commentary on ddRNAi and the Gu et al paper.

We've seen very little in terms of therapeutic programmes against CNS diseases such as Parkinson's.

U-Penn researchers have published a paper this week with a breakthrough in that misfolded a-synuclein protein actually spreads from neuron to neuron, & not spontaneously appearing across neurons.

Do these advances, and the recent advances in AAV vector delivery, get us to the point where a clinical programme using shRNA-SNCA at lower dosage and improved targeting is a real possibility?

Anonymous said...

"If the HepC program that Benitec acquired from Pfizer was actually working, do you think Pfizer would have let it go??"

Yes, they let a lot more go than just the HCV programme, of which sharp bio entrepeneurs have already snapped up. Pfizer execs who cut their R&D arms off wouldn't even be able to tell you what they let go.

Fact is with respect to the HCV TT-034 programme, the Pfizer people who didn't get sacked, did not want to let it go. It took Tacere 9 months to convince them otherwise, so the programme could get going again.

So despite the critism, TT-034 is now back on track toward the clinic. Smart move by Benitec.

So assumptions can be costly. Check this article regarding Pfizer's Sandwich R&D cuts. They've already started buying back into their old programmes.


Dirk Haussecker said...

I'm curious whether HepC will actually go into humans with Benitec. HepC clinical programs have historically been very expensive...I believe telaprevir should go down in biotech history as the most expensive program for a drug to get first approval.

Finally, part of the reason why Benitec had to buy back HepC is because all their own outsourced programs never came anywhere close to the clinic, despite bold promises otherwise. I cannot see any evidence that Benitec's strategy over the last 2 years has worked. The licenses...apparently no cash consideration involved. What does this indicate?

Anonymous said...

As I understand it many applications of ddRNAi will require the use of multiple cassettes, HCV, HBV, HIV, and this technology in patented by BLT and the patent extends to 2025. If this is the case then the all is not lost in terms of BLT's extensive patent position. The is also the possibility that the core Graham patents could have their life extended by the USPTO.

Anonymous said...

To be fair, BLT did pay for City of Hope's HIV clinical trial. BLT are hindered due to geography, non-existant government grants and an all-time low risk appetite for biotechs. They cannot re-establish a lab because of lack of cash, partly exaccerbated by the research exemption which dried up it's only source of revenue and partly due to patent interference. A terrible state of affairs really for a company that has/had such great prospects. A merger between Gradalis and BLT could quite easily solve this problem, with David Shanahan being both CEO of Gradalis and President of his own non-profit research centre, dramatically reducing the costs of early stage clinical trials. This would also prevent patent interferance by BLT or royalty payments/milestones to them should Gradalis want to commercialise it's cancer drugs.

Anonymous said...

Nearly 100 people were working on this program, hundreds of animals tested, 100% transfection with no toxicity issues after 180 days. Minimum $30 million spent, including the upfront to Tacere. Benitec gets the new improved version, ready for Phase I/II, for $1.5 million worth of shares after selling it to Tacere in the first place. Worth a punt I reckon.

Sounds to me like Pfizer threw the baby out with the bath water.

Anonymous said...

I believe Benitec bought the Hep C program because they got a great price and had a great relationship with the Tacere folks. Weren't they former Benitec employees spun out in 2006 during the bleak years of the patent war?

I think they are pursuing a great strategy. Maybe they passed on upfront cash for a higher royalty rate or a higher milestone payment? Who really knows? They are the only ddRNAi platform company in the industry. This has to give them a tremendous lead and competitive advantage even after the patents expire. Are their patents good through 2025?

Anonymous said...

Usher Syndrome Gene Therapy Safe Thus Far In Three Patients

Nov. 20, 2012 – Oxford BioMedica, a leading gene therapy development company in the United Kingdom, has received a positive interim safety review of its ongoing Phase I/IIa clinical trial for the UshStat® gene therapy for people with Usher syndrome type 1B. The first three patients experienced no adverse events for up to six months after treatment. The positive review by the study’s Data Safety Monitoring Board, an independent panel of ophthalmology and gene therapy experts, enables the company to treat additional patients at a higher dose level.

Anonymous said...

I can't wait until Benitec/CSIRO start targeting agricultural and veterinary applications of ddRNAi.

Anonymous said...

From Tacere publication, the AAV formulation for HCV is only for single shot, although claim to last for a while but certainly is questionable the formulation is ever going to reach the clinic. Benitec must have a good reason to issue more stock to absorb Tacere.

Anonymous said...

Why do you say it is questionable it will ever reach the clinic?

Anonymous said...

Dirk said because of the high cost to run an HCV trial. He doubts Benitec could afford it. At market cap of $15M he's right. So they'd have to partner this with a big pharma.

The good news is if they are impressed enough with the TT-034 pre-clinical they will pay whatever needed to run HCV trials.
It's only the hottest target in big pharma.

With it would come opportunity to licence on the HBV programme, where the big hurdles already would have been cleared with the HCV programme.

HBV and HVC treatment go to shop being the ultimate prixe. Not totally impossible as TT034 non human primate study results were very impressive. That's why the pfizer people working on it, the ones who actually knew, didn't want to let it go.

Anonymous said...

I can see why the Pfizer people didn't want to see it go. 100% transfection with no toxicity for 180 days. I bet some Pharma will pay for the trials. Benitec has nice proximity to Asia where I believe HCV and HBV are significant health problems.

Lots of good things happening for Benitec now. Finally. The science is great and I see a bright next 15 years for them.

Anonymous said...

Regarding TT-034. How much is a Phase I/II clinical trail going to cost when the compound has already been manufactured and is a one shot treatment?

Anonymous said...

Let us see how the Benitec (Tacere) HCV program develops, we certainly hope the best. Without an effective pre-clinical model, it is hard to predict whether the treatment will reduce the viral load in man, particularly if one can only do one shot. Viral escape is well known. Risky venture. Hope they get support, but I doubt it will attract big-pharma.

Anonymous said...

$5-$10M should complete a Phase I/II, so I don't see why bigpharma need to come to the rescue. A fully-underwritten capital raising will raise this amount, no problem. Benitec have stated they are not particularly interested in partnering until Phase I is completed. Funny thing is, Pfizer could take out BLT, including all their programs and IP, for well less than the $145 million collaboration deal they originally had with Tacere. If TT-034 is successful in Phase I, they will have to pay at least 10 times that just to get that one drug back.

Anonymous said...

a one shot, which this is, is going to be less complicated than current HCV trials. Viral escape, well in the long run ddRNAi multi-cassette approach targeting multiple conserved regions sounds elegant compared to the current small molecule concoction approach.

ddRNAi, particularly multi-cassette approaches, is shaping to be the perfect therapeutic complement to genome sequencing of disease.

where are the advances in genome sequencing going to stop? think of moore's law.

Biotech, personalised medicine, and digital revolution converge. An outpatient day procedure, upload a pic whilst you get your cancer, HIV or HCV one shot, and twitter it to your mates.

sounds far fetched,and the above scenario is still a long way to go, but regardless it's already starting to happen.

for the first generation have a look at the cancer trials at Mary Crowley Centre for Cancer Research on youtube.


Anonymous said...

Dirk, re: biomics sirna topical application. here's the Molecular Therapeutics paper on it.


"Topical application of siRNA formulation significantly lightens brown facial hypermelanosis and lightens normal skin in Asian individuals. This treatment represents a safe and effective therapy for melasma, suggesting that siRNA-based agents could be developed for treating other diseases such as melanoma."

maybe it's just a matter of Biomics commissioning OxTerms to give their good work credible translation.

Anonymous said...

FYI AMT did go out of business but was taken over by uniQure - including RNAi research. The company recently received the first EMEA approval for their (non-ddRNAi) gene therapy product

Anonymous said...

on the same lines as Biomics --> Sirnaomics?!?!

Anonymous said...

Calimmune's Gene Therapy HIV trial has FDA approval for a start.

next step is recruiting.


Dirk Haussecker said...


Anonymous said...

More progress in yet another ddRNAi program:


Who needs a lab when you have lots of co-labs??

Anonymous said...

Dirk, what do you make of Somagenics decision to wait and see what happens in the HCV market before proceeding with a clinical trial?

Do you think Benitec should take note of this decision when considering their TT-034 program?

Dirk Haussecker said...

Don't know about Somagenics but, yes, the HCV market is very dynamic and it will be a challenge to find a niche, esp. if you are non-oral. But putting the HCV program on ice would look bad on Benitec after just having brought the program back in-house.

Anonymous said...

Dirk FYI re Somagenics


Robert Zack said...

Benitec is in the clinic.

Anonymous said...

...and down the gurgler.

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