Antisense Therapeutics Dressing Up with RNAi Delivery

The boundaries between RNAi Therapeutics and Antisense Therapeutics have become blurrier.  Originally, the basic practical difference between RNAi and antisense was as follows: on the one hand, RNAi is very powerful, but it requires special formulation chemistry which often is the tox-limiting factor and necessitates that the drug be administered intravenously.   On the other hand, antisense can be utilized in unformulated/naked form and be administered subcutaneously, but it is less potent and involves the accumulation of large, problematic levels of phosphorothioate molecules in a range of tissues, primarily the kidney, liver, spleen, and probably the vasulature.

   

Recently, however, some RNAi approaches have become more antisense-like in terms of patient convenience in that conjugate technologies and ‘self-delivering’ RNAi triggers that can be administered subcutaneously have advanced into the clinic.  Over the last two weeks then, two pieces of evidence emerged of the reverse trend of antisense technologies adopting delivery technologies originally developed for RNAi Therapeutics.  The is done for improved potency and could also bring critical safety advantages.

Evidence 1: ISIS and Alnylam Find SNALP Delivery to Increase RNaseH Potency by ~20-fold

In a paper by Prakash et al. in ACS Chemical Biology, scientists from ISIS Pharmaceuticals and Alnylam demonstrate significant potency enhancements for RNaseH antisense and single-strand RNAi to be gained from the use of Tekmira’s SNALP technology.  As a reminder, the two companies once collaborated on single-strand RNAi before Alnylam terminated that relationship with ISIS continuing work on the subject, so the present publication relates to historical work during their partnership, but is relevant to ISIS' continued efforts.

The study simply compared the knockdown potencies of RNase H gapmers (phosphorothioate 2’ MOE chemistry), single-strand RNAi triggers (heavily modified), and dsRNAi triggers (ssRNAi molecule as guide together with complementary unmodified passenger strand), either as unformulated oligonucleotides or when formulated with SNALP containing the DLin-KC2-DMA lipid.  This lipid, developed by Old Tekmira (see Semple et al. 2010, Nature Biotech), was a critical ingredient of the first ‘2^nd-generation’ SNALP showing significantly improved liver knockdown potency over the original DLin-DMA SNALPs. 

When targeting PTEN in mouse livers, none of the oligonucleotides alone exhibited knockdown activity at the 4.5mg/kg dose tested.  Only when increasing the dose up to 100-200mg/kg did the RNaseH gapmer and ssRNAi trigger exhibit marked knockdown.

   

In stark contrast, profound knockdowns were achieved with SNALP-formulated RNaseH gapmer and the ssRNAi trigger at the 4.5mg/kg dose.  Remarkably, for the RNaseH gapmer, the activity was obtained at liver oligonucleotide levels that did not differ much from that of the unformulated oligonucleotide (which did not exhibit PTEN knockdown).  This means that SNALPs not so much increases the delivery to the liver per se, but the functional delivery of RNaseH gapmer, possibly by liberating phosphorothioate oligos along the endosomal cellular uptake pathway.  This finding also makes it difficult to believe some reports that phosphorothioate oligos administered without special formulation accumulate in the nuclei as this is the site where they are supposed to be mostly active.  Where the non-functional oligos actually end up is an important question as this also relates to the safety of these molecules.

Overall, the study shows that formulation with RNAi delivery technologies can greatly enhance knockdown potency.  It should be added here, however, that this single-dose study somewhat underestimated the potency of RNaseH antisense as normally a loading dose schedule would be performed to reach threshold tissue levels required for knockdown activity of unformulated PS-RNaseH gapmers.

The lower dosages and more targeted delivery with RNAi delivery may also reduce the typical toxicities associated with phosphorothioate antisense.  Indeed, one may even be able to do away with the phosphorothioate modification altogether.  Of course, toxicity related to the delivery vehicle also needs to be considered, but the authors noted in their paper that SNALP was ‘well tolerated’ and that no changes in liver enzymes or in body weight loss were seen.

To further gauge the value of SNALP and RNAi delivery in general for gapmer antisense technology, a more detailed time-course analysis of the knockdown and the tissue concentrations would be useful.

Evidence 2: Regulus Therapeutics to Employ Alnylam GalNAc-Conjugation for Mir-122 HCV Program

In another example of the value of delivery technology originally developed for RNAi Therapeutics for single-strand oligonucleotide Therapeutics, Regulus Therapeutics disclosed at last week’s Needham conference that it would use Alnylam’s GalNAc conjugation for the delivery of anti-miR122 in its HCV program which is to enter clinical development in 2014.

With this, Regulus expects to achieve once-monthly-dosing with their single-stranded anti-miR at lower doses (0.3mg/kg) than the weekly LNA-based phase II compound by competitor Santaris (>1mg/kg).  Importantly, Regulus claims that HCV patients go to see their doctors to check their antiviral responses on a monthly basis anyway.  Consequently, these routine visits for patients on anti-HCV therapies could then be combined with the subcutaneous administration of the anti-miR.  This obviously would increase compliance in an era in which oral HCV meds reach high cure rates in clinical trials, but the actual cure rates in the field are significantly lower due to rate-limiting compliance issues (h/t to John Alan Tucker from Zacks Investment Research).   

Without showing the data, it remains to be seen whether and by how much GalNAc conjugation improves anti-miR potency.  The once-monthly dosing, however, indicates that once the single-stranded oligonucleotide is in the cytoplasm (where the target is), it is trapped and stays there for quite some time instead of being washed out again into the interstitial space (outside the cells) which would be the case if extracellular and cellular oligonucleotides were in dynamic equilibrium.     

It will be interesting whether the GalNAc approach will utilize the phosphorothioate backbone which ISIS Pharmaceuticals is using at all.  If not, the decision would indicate that ISIS satellite company Regulus is concerned about the safety implications of that chemistry, especially in patients where the liver is already diseased.

Get It Today...The RNAi Therapeutics Investment Guide for 2013

What is driving the RNAi Therapeutics market, where are the pockets of value creation, and where are the risks?

The 'RNAi Therapeutics Investment Guide 2013' will answer these questions and more.  The Guide should be particularly useful for the following audiences:

1) Drug developers which consider investing in RNAi Therapeutics drug development;

2) Drug developers already engaged in RNAi Therapeutics development to help position themselves in the competitive market place;

3) RNAi Therapeutics investors, especially those in the public markets to understand the market place dynamics and to help identify investment opportunities.

42 pages.

Table of Contents                                                        

                                                                               

Abstract                                                                                 

Table of Contents                                                                   

1. The RNAi Therapeutics Opportunity                                 

2. RNAi Therapeutics History                                               

            A. The Geek Phase (2002-5)                                    

            B. Irrational Exuberance (2005-8)                             

            C. Doubts and Despair (2008-11)                              

            D. Revival (2011-present)                                        

3. RNAi Therapeutics in 2013                                                

A.    State of the (RNAi Delivery) Art                                     

                        i. SNALP                                                        

   ii. Dynamic PolyConjugates                           

                        iii. GalNAc-siRNA Conjugates                      

                        iv. Self-delivering RNAi Triggers                   

                        v. Viral Delivery                                             

B. The RNAi Trigger IP Landscape in 2013             

C. Sector Tailwinds                                                    

  i. RNAi Therapeutics Hits Sweet Spot of Secular Drug Development Trend                                                                      

            ii. RNAi Therapeutics as Part of Oligonucleotide Therapeutics Resurgence

                                                                                    

D. RNAi Therapeutics Deal Dynamics in 2013          

E. Clinical Trial Catalysts in 2013                              

            i. TKM-PLK1                                                 

            ii. ALN-TTR02                                               

            iii. ALN-TTRsc                                               

            iv. RXI-109                                                     

            v. ARC520                                                      

            vi. Cal-1                                                          

4. Potential Pitfalls                                                                  

5. Outlook Beyond 2013                                                        

6. Company Profiles                                                               

A.    Publicly Traded Pure Plays                                   

                        i. Alnylam                                                       

                        ii. Arrowhead Research                                  

                        iii. Benitec                                                       

                        iv. Bioneer                                                       

                        v. ISIS Pharmaceuticals                                  

                        vi. Marina Biotech                                          

                        vii. RXi Pharmaceuticals                                 

                        ix. Silence Therapeutics                                  

                        x. Tekmira Pharmaceuticals                            

B.     Big Pharma Players                                               

                        i. Merck                                                           

                        ii. Novartis                                                      

                        iii. Takeda                                                       

                        iv. Other Big Pharmas, Including AstraZeneca          

C.     Privately Owned Pure Plays                                             

           D. Other RNA Therapeutics Companies and Technologies   

Extra: The Business of RNAi Therapeutics 2013 (slide presentation).

Extra: Presentation slides.

Keywords: Alnylam, Arrowhead Research, Benitec, Marina Biotech, RXi Pharmaceuticals, Silence Therapeutics, Tekmira, Merck, Novartis, Big Pharma, SNALP, DPC, GalNAc, conjugates, self-delivering RNAi triggers, Dicer-substrates, nucleic acid modifications, TTR amyloidosis/ALN-TTR, HBV/ARC520, PCSK9/hypercholesterolemia, PLK1/cancer, RNA Therapeutics, antisense-mediated gene knockdown, intellectual property, orphan diseases, liver, safety.

Pricing

Drug development companies up to 10 employees: 250 Euros/300 US dollars

Drug development companies over 10 employees: 500 Euros/600 US dollars

Other companies (e.g. financial institutions and investment funds): 400 Euros/500 US dollars

University libraries: 95 Euros/120 dollars

Individuals (private use only): 95 Euros/120 US dollars

Prices exclude 19% VAT for German customers (corporate and non-corporate) and non-corporate customers in the European Union.

Payments either by bank transfer or paypal.

When ordering, the following information is required:

Full name and address

Use (corporate, individual etc)

Billing name and address if different

Order by contacting me at myfirstname.mylastnameATgmail.com
My first name: dirk; my last name: haussecker

RNAi Therapeutics Companies Cashed Up and Ready to Strike

With the $36M fund-raising last night, Arrowhead Research has become the 4^th publicly traded RNAi Therapeutics company in short succession to cash up for a real shot at drug development success.  The other companies are Tekmira, RXi Pharmaceuticals, and as it was made official this morning, Silence Therapeutics.

The money will be spent on promising drug candidates in the cancer field (TKM-PLK1, Atu027), chronic HBV (ARC520), and scarless wound healing (RXI-109) without having to take dangerous short-cuts or partner the crown jewels prematurely.  Moreover, it will help to advance critical delivery technologies such as DPC, SNALP, and self-delivering RNAi triggers which should provide for non-dilutive partnering opportunities without having to entertain low-ball offers.   

It is notable (and not surprising) that unlike Marina Biotech and Benitec which so far have failed to similarly escape the financing maelstro, Tekmira, Arrowhead and Silence all sport healthy in-house R&D operations.  RXi does not have such operations- yet.  However, they were critical for bringing RXI109 into the clinic on which the company’s ~$80-90M market cap rests.   

The cash infusions should also give the companies, which among them own most of the leading platform technologies, the opportunity to make up for the 20-30 fold gap in valuation to the likes of ISIS Pharmaceuticals (2.3B market cap in antisense), Alnylam (1.5B market cap in RNAi), and Sarepta (1B market cap for a phase II orphan indication).  If monoclonal antibodies are any guide, this will not play out as the winner-takes-all the public markets have it right now.

Below is a quick rundown of the newly cashed-up crop of RNAi Therapeutics companies for those new to the field.

Disclosure: I am long Tekmira and Arrowhead Research. 

Disclaimer: Investments in RNAi Therapeutics carry more risks than you want to know and think about.  The following can only be a very brief teaser for you to start your own due diligence.

Arrowhead Research: In the pursuit of a cure for chronic HBV

Market cap: ~60-70M (of which ~$35M in cash)

Unless Arrowhead Research comes up with a compelling orphan drug application of its technology when it will disclose its second development program in H1 2014, investment success will hinge on the fate of ARC520.  ARC520 is Arrowhead's unique attempt at a functional cure for chronic HBV that will enter clinical development shortly.  I interpret last night’s fund raising in that the IND-enabling tox studies were positive.  The results of these will be critical in determining whether ARC520 can be dosed high enough to safely achieve the type of 90% HBsAg reductions that will be required for that lofty goal.

With cash of around $35M, Arrowhead can now retain full rights to that program at least after value-inflecting phase I proof-of-concept knockdowns have been achieved.  To extend the cash runway beyond the predicted ~2 years, Arrowhead owns the exciting Dynamic PolyConjugate delivery technology, particularly the subcutaneous version and potential new developments for cancer applications.  DPC offers an obvious non-dilutive funding opportunity.

Having slept over the announcement of the PIPE financing which doubles the share count near multi-year lows, I believe it still was a good one in a difficult situation.  Potential partners would have liked to exploit the financial weakness of Arrowhead and the fast-money crowd (think Dawson and Rodman) would have liked to reap risk-free gains from trading around a public offering.  Instead, Arrowhead seems to have found one or more significant investors that share the vision with management.  It is comforting that besides the sweet entry price, no special gifts in the form of warrants and otherwise seemed to have been handed out.  No wonder Arrowhead closed up 13% the day after the financing.

Upcoming catalysts:

  - Determination of maximally tolerated dose in volunteer study with ARC520;

-          - DPC partnering (always on the table);

-          - Modest ALN-TTRsc results would emphasize value of DPC (mid-2013) and may get Alnylam into  play;  

-          - DPC data on cancer (probably H1 2013);

-          - Nominating new development candidate (Q2 2014);

-          - ARC520 viral knockdown results in the Hong Kong trial (H1 2014).

Tekmira Pharmaceuticals: Quality Technology, Quality Financial Management

Market cap: ~$70M (of which ~$40M in cash)

If you are looking for relative stability in RNAi Therapeutics, Tekmira could be attractive.  It was its SNALP technology that enabled the current revival (with well over $300M capital inflows in the industry in the first 4 months of this year) and remains one of the two leading systemic delivery technologies with the value possibly shifting towards oncology indications.  Its leading development candidate, TKM-PLK1 for solid cancers, is about to enter phase II studies and has yielded promising dose-related evidenceof efficacy in phase I. 

The company’s aerosolized LNPs could be an unanticipated windfall for investors, although I have yet to see the all-important safety data.  Its Ebola biodefense program is not valued much by the market, but has been an important stabilizing factor for the company in terms of funding and represents an attractive commercial opportunity in its own right.  

Upcoming catalysts:

-          - TKM-PLK1 data from the expansion cohort (H2 2013);

-          - Design and initiation of phase II studies for TKM-PLK1 (H2 2013);

-          - Follow-up candidate and potentially data for TKM-EBOLA (H2 2013);

-          - Nomination of new development candidate;

-          - $5M milestones each if ALN-TTR02 by Alnylam enters phase III development and Ascletis   initiates a trial in China with ALN-VSP02 for primary liver cancer (both H2 2013/H1 2014);

-          - Partnering of SNALP delivery technology, also for non-RNAi, non-therapeutic applications (always on the table);

       - Monetization of Talon royalties (uncertain timing if at all) 

Silence Therapeutics: A novel cancer approach and strong purchasing power

Market cap: ~150M (of which $30-35M in cash)

Following a remarkable financial turnaround and management reshuffle, Silence Therapeutics has become a force to be reckoned with once again.  Unlike in the early days, it is the product candidates, and not RNAi trigger IP, that is the focus of the New Silence.

First and foremost is Atu027, an endothelially targeted RNAi Therapeutics aimed at preventing cancer metastasis.  The company believes that Atu027 will be most useful in combination with small molecules that aim at killing primary tumor cells and consequently is about to initiate phase Ib/phase II combination trials.

According to the information in the latest financing (which was approved today), a significant investment will be made in building a broader pipeline.  This will take advantage of the company’s three lipid-based delivery technologies: Atuplex (broad endothelial cells), DACC (lung endothelial cells), and DBTC (cells in the liver).  In general, the market cap and cash infusion position the company well to pick up some distressed RNAi and other assets.

Upcoming catalysts:

-          - Initiation of pancreatic cancer combination trial with Atu027 (H2 2013);

-          - Nominating new development candidate(s) (H2 2013);

-          - Pre-clinical data on liver delivery technology (2013);

-          - Partnering, also for non-RNAi, non-therapeutic applications

RXi Pharmaceuticals: Just dermal anti-scarring no more  

Market cap: $80-90M (of which ~$20M in cash)

RXi Pharmaceuticals is the company that popularized the self-delivering RNAi trigger concept.  Unfortunately for the company, it made a critical IP mistake of staying clear of the most potent versions it could have created with its ideas (à dsRNA length).  Furthermore, it seemed that it never was able to get anything into the clinic.

This changed last year with phase I initiation for RXI-109, a potentially superior alternative to Pfizer’s antisense-based dermal anti-scarring candidate.  However, as part of a reorganization, it essentially got rid of its R&D to focus all resources on RXI-109.

As you know, getting rid of in-house R&D is a mortal mistake if you want to be a platform-based company.  With the recent $16.4M cash infusion by OpkoHealth it will be interesting to watch whether they can become a self-delivering RNAi trigger platform-based candidate again, or whether the claimed company growth will largely be around expanding the indications of RXI-109 to other anti-fibrotic indications, especially ocular ones.

Upcoming catalyst:

-          Presentation of phase I data from RXI-109 (any day).

Mirna Therapeutics Brings First MicroRNA Replacement Therapy into Clinic

It has been a long wait, but 5 years following the initiation of anti-miR122 development for the treatment of HCV infection by Santaris, a second microRNA Therapeutics has now entered clinical development.  MRX34 by Mirna Therapeutics is a miR-34a replacement therapy for the treatment of liver cancer or cancers with liver involvement.  

MicroRNA replacement therapy is technically very similar to RNAi Therapeutics.  It involves adding to cells naturally occurring microRNAs to orchestrate typically a range of processes deemed to be therapeutic.  It is added to cells in the form of double-stranded RNA triggers very much alike RNAi triggers and can therefore largely utilize the same types of delivery approaches.  The delivery technology for MRX34 is the NOV340 SMARTICLE technology owned by Marina Biotech.  This liposomal delivery technology is distinct from SNALP, most notably by employing ionizable ‘amphoteric’ lipids, lipids that can take on both positive and negative charge depending on pH, and not ionizable cationic lipids, lipids that merely become positively charged at acidic pH, as in the case of SNALPs. 

An extensive literature supports miR-34a as an exciting microRNA for replacement therapy in cancer rivaling the most famous let-7.  Accordingly, miR-34 emerged as Mirna Therapeutics’ top priority based on extensive screening conducted at Ambion (now Life Technologies) from which Mirna Therapeutics was spun out.  Under transcriptional control by the guardian of the genome, p53, its pleiotropic effects range from cell cycle inhibition, to counter-acting anti-apoptotic mechanisms and to sensitizing towards chemotherapy.  The choice of liver-related cancers was thus not based on cancer biology (many cancers cancers would apply), but largely a function of where the company thinks NOV340 can deliver to.

This to me raises again the question of whether liver cancer is closer to normal liver or whether it is closer to solid tumors in general in terms of delivery.  MRX34 thus follows Alnylam’s reasonings as manifested by ALN-VSP02, but goes against what is known about blood supply differences between normal liver and liver cancer and what is practiced by the likes of Tekmira and Dicerna.

The phase I study will be a typical dose-escalation study seeking to determine the maximally tolerated dose.  Given the importance of delivery, close attention should be paid to the pharmacokinetic and biodistribution data from this trial.  These should start to become available in the first half of 2014.

The development is positive for at least two other companies.  As the provider of the delivery technology, Marina Biotech obviously stands to gain financially and otherwise from such clinical milestones.  Whether it will be sufficient to pay off its debts that have come due and consequently avert bankruptcy is an open question.  Due to the financial distress, Marina Biotech struggled to enter into deals giving it fair compensation for its technology.

Although the most direct competitor to Mirna Therapeutics, InteRNA as the other major microRNA replacement company in oncology should also benefit from the initiation of the phase I study as it helps to validate its approach.  The question will be whether among its stable of microRNA replacement candidates, there are some with as robust activities as miR-34a.   

BMS Partners with Santaris

In other recent news related to microRNA Therapeutics, Big Pharma Bristol-Myers Squibb partnered with LNA antisense company Santaris under which the Danish company collected $10M upfront.  Although the press release did not specify much the aims of the the alliance, referring to RNA Therapeutics broadly, a few factors speak in favor of microRNAs being involved.  

Most notably, BMS had terminated an phosphorothioate RNaseH antisense collaboration with ISIS Pharmaceuticals.  As Santaris in turn had terminated their PCSK9 phosphorothioate RNaseH antisense program as well, most likely due to kidney toxicity (--> phosphorothioate chemistry as also in Prosensa; van Poelgeest et al. 2013), the first suspicion that BMS sought out Santaris as a more potent PCSK9 alternative becomes less compelling.  Moreover, ISIS Pharmaceuticals is suing Santaris over US patent infringement of RNaseH technology which should hinder the ability of Santaris to enter into relationships with US companies for RNaseH antisense purposes.  Of course, the deal could also indicate that a settlement is in the making...

Let’s Go to San Francisco: Trends from the 4th RNAi Research and Therapeutics Conference

Still looking for an interesting RNAi Therapeutics conference to attend? The 4^th RNAi Research&Therapeutics conference to be held in San Francisco June 20-21 may be a good choice to satisfy your cravings for not only RNAi Therapeutics research and development, but also the wider applications of RNAi gene silencing for target identification and new interesting trends in targeting non-coding RNAs.

Register with discount code ‘RNABLG13’ for 20% discount and free RNAi Therapeutics blog T-shirt here.

Reading through the abstracts of conferences like this one is helpful in understanding the trends in a scientific field.  As an appetizer, the following will highlight a few of those promising scientific directions, challenges, and future investment flows as seen through the lens of an RNAi Therapeutics nerd.

Delivery, delivery, delivery

An old headline, but as the key value-driving effort, delivery naturally is a focus of an RNAi Therapeutics conference.  Importantly, the two most advanced and promising systemic delivery technologies, SNALP/LNPs and DPCs, are represented.  Pieter Cullis, an academician with ties to the Tekmira, AlCana, and Alnylam love triangle, will be talking about Tekmira-type LNPs.  Interestingly, while these LNPs are known to have applications for knocking down genes in the liver, solid tumors, and potentially sites of inflammation and lung epithelia in aerosolized form, the abstract notes their use in the central nervous system (CNS) for serious neurological disorders.  Of course, the CNS is the organ with many of the major unaddressed medical needs, so I am curious whether there will be actual data showing good distribution and efficacy of LNPs in this organ.  While I consider the CNS challenging in terms of achieving wide distribution with direct administered nanoparticle-based delivery technologies, the immune challenges of nanoparticles, including complement activation and phagocytic uptake, might be lessened in this organ.

David Lewis from Arrowhead Research will present the latest about the exciting DPC delivery technology, including data from the chronic HBV program (ARC520).  By then, IND-enabling tox studies for ARC520 should be completed and the first DPC-related IND filed.  His talk should also be of interest to those not as obsessed about DPCs as I am as he will speak about some of the mechanistic insights of the key endosomal escape process, insights which should be more broadly applicable.

Trinna Cuellar from Genentech will be speaking about ‘A systematic evaluation of antibody-mediated siRNA delivery and silencing’.  It seems that Genentech hopes to create RNAi Therapeutics value based on its expertise in monoclonal antibody pharmacology.  The talk should elucidate whether the strategy envisions only minimal antibody-siRNA conjugates, or whether it further embraces other delivery platforms.  Of note, Tekmira once mentioned that it had a technology evaluation with Genentech on antibody-targeted SNALPs and made the bold prediction that ‘Roche will be back’.

Curiously, not one but three presentations concern aptamer-targeted RNAi trigger delivery (note: aptamers are antibody-like nucleic acids).  We have had a number of reports of such aptamer-siRNAs, especially for cancer and HIV infection.  However, these reports have encountered much disbelief, especially since they lacked an explanation for their postulated endosomal escape.  With credible knockdown activity reported with the GalNAc-siRNA conjugates by Alnylam, aptamer-RNAi triggers should be similarly gaining in credibility, especially when combined with self-delivering RNAi trigger chemistries.  An experiment that I would like to see as part of the field regaining credibility is an ASGPR-targeted aptamer-RNAi trigger conjugate (ASGPR is the receptor targeted by the GalNAc sugar).  In addition to building confidence in the approach, it would also be an alternative to GalNAc-RNAi triggers should the sugar moiety cause injection site reactions in humans.

Some of the aptamer-RNAi trigger presentations also touch on the subject of bifunctional RNAi Therapeutics, meaning RNAi Therapeutics formulations with a second non-RNAi therapeutic activity.  These secondary activities may include receptor blocking or toll-like receptor (TLR) activation as in a talk on CpG-STAT3 siRNAs by Hua Yu from the City of Hope.

In ddRNAi it is about the RNAi trigger design

The conference also includes presentations on microRNA processing and their implications for ddRNAi trigger design.  Unlike in RNAi Therapeutics induced by synthetic RNAi triggers, RNAi trigger design remains a crucial value-creating activity in DNA-directed RNAi Therapeutics.  Unfortunately, its importance has been entirely lost from the commercial field (if you really want to aggravate me, show me a U6-driven shRNA being put into clinical development).  Fortunately, academic efforts mean that ddRNAi trigger design continues to advance.

In his Keynote address, Mark Kay from Stanford University will present insights from his lab on microRNA processing and, crucially, how that knowledge can be put into practice for ddRNAi Therapeutics.  Especially recent work from Shuo Gu in his lab on Argonaute-specificity, strand selection, and Dicer processing have advanced this field and should also provide for fresh ddRNAi IP opportunities.  A presentation by Marco Weinberg from the Scripps Research Institute meanwhile is focused on an earlier step in microRNA processing, the primary microRNA recognition and cleavage by the Drosha enzyme.  ddRNAi trigger cassettes requiring the Drosha processing step promise to be more natural and cell-specific alternatives.  

MicroRNAs hot, lncRNAs getting hot

Functionally closely related to RNAi, it is not surprising that the conference program includes microRNA Therapeutics.  As by far the most advanced microRNA Therapeutics program, the update on the anti-miR122 program by Santaris for HCV infection (phase II) will be a highlight for those that have not been paying close attention to the results.  For those more familiar with the story, it will be of interest how Santaris aims to position this innovative treatment approach in an increasingly crowded and competitive field.

There will also be presentations on long non-coding RNAs (lncRNAs) and their therapeutic use.  LncRNAs are RNAs that do not code for proteins and are usually expressed in between protein-coding genes or antisense to or just upstream of protein-coding genes.  As more and more examples of such functional non-coding RNAs emerge, a whole cottage industry has emerged to cater to them.  Interestingly, reports suggest that lncRNAs can often be suppressed by RNAi triggers.  This is a departure from the classical thought that RNAi triggers can only target protein-coding mRNAs.  Similar to microRNA antagonists, targeting lncRNAs that silence other genes, can activate gene expression.  Oligonucleotide research reagent companies have naturally recognized the commercial potential of this by offering large lncRNA-targeting RNAi trigger libraries and Zaklina Strezoska will present the design approach by Thermo Fisher.

RNAi trigger libraries are also the focus of presentations by Michael McManus from UCSF and Iain Fraser from the NIH.  These researchers employ the libraries for understanding biological processes, an effort that is also suitable for new drug target identification.  The library approach by McManus is unique in that every gene is targeted by many (~25) shRNAs.  This is done to increase the confidence that hits concern real targets and not just some off-target noise.  The approach is critically facilitated by next-generation sequencing (NGS).

Next-generation sequencing is also the focus of a late afternoon session at the conference.  Since NGS is ubiquitous in today’s research, keeping up-to-date on technological advances in this field is part of the homework of any RNAi Therapeutics buff.

The conference will finish on a Friday.  If you like to digest all the information and be inspired by nature at the same time, I recommend that you stay the weekend and rent a car to see some redwoods, coastline, and villages inhabited by eccentrics north of the Golden Gate Bridge.  Good weather guaranteed.

Register with discount code ‘RNABLG13’ for 20% discount and free RNAi Therapeutics blog T-shirt here

FDA Proactively Questions Eteplirsen Biomarker Methodology

Orphan drugs tailored to address defined genetic lesions account for a significant portion of the current value of RNA Therapeutics.  One of the reasons is that they promise much more economical approval pathways, including shorter timelines, smaller patient populations, and biomarker-based approval.  Following RNAse H antisense Kynamro by ISIS Pharmaceuticals/Genzyme Eteplirsen for homozygous FH, eteplirsen by Sarepta for Duchenne Muscular Dystrophy (DMD) offers us another front-row view of this process.

Currently, the question on most observers' minds is whether the biomarker data gathered so far, most notably from a phase IIb trial in just 12 patients is sufficient for accelerated approval (AA).  Towards this end, Sarepta and the FDA had a meeting a few weeks ago to discuss whether the FDA would accept for review such an AA submission.  According to the Minutes of the meeting which were discussed in a press release by Sarepta on Monday, the FDA does not believe the data it has seen warrants an AA submission and asked the company to come back with more data.

I am one of the very few open critics of eteplirsen and have explained my rationale why I have little confidence in the phase IIb data that have catalyzed much popular support: a manipulated trial conduct where essentially all the supportive evidence was collected after unblinding of the trial and a cherry-picking way of presenting them.

What emerged from the recent FDA interaction appears to be largely consistent with my concerns.  Today, I would like to highlight a critical issue when it comes to pursuing biomarker-based approval strategies and that was the focus of the recent interaction.

How you measure biomarker is as important as the biomarker per se

It does not reflect well on a company seeking accelerated approval when, after two decades or more of morpholino-based drug development, it has yet to establish sufficient manufacturing capacity to satisfy even the small patient population that would be eligible for eteplirsen.  The same poor planning becomes evident when the FDA appears to be questioning the method by which the biomarker (dystrophin) data was collected: what is the value of a biologically strong biomarker if you can’t reliably measure it to draw comparisons?

The dystrophin expression by immunofluorescence and Western blot were from biopsy samples.  Obviously, muscle tissue is everywhere in the body and any drug efficacy will vary depending on the muscle.  Even within a given muscle, (revertant) dystrophin expression is known to be variable in DMD patients, so despite of consistently taking biopsies from the same muscle before and after treatment, you may not be able to tell 2 or 3-fold differences.  This problem was noted by the investigators in the first phase II trial of eteplirsen in discussing the discrepancies of Western blot and immunofluorescence data.

Of course, this sampling issue could be addressed with large patient numbers, but not with the 12 as in this trial.  Given the importance of methodology, it is surprising that Sarepta missed the opportunity to provide the FDA with sufficient related information to be in a position to more conclusively tell whether it’s worth submitting for AA or not.

Issues the FDA might want to know more about could be the selection strategy of biopsy location and whether the antibody was appropriately chosen also for taking into account revertant fibers.  Revertant fibers are a phenomenon due to either secondary mutations or alternative splicing which leads to dystrophin expression in many DMD patients.  This might also shed light on for example why after 12 weeks on drugs, no dystrophin expression was seen, but at 24 weeks it was. 

Another important question that the detailed methods might answer is the quantitation of the absolute amount of dystrophin expression (not percent fibers expressing).  Based on the phase II study publication, it appears to be quite difficult to do even just semi-quantitative calculations on fuzzy Western blots.  Of course, once the real expression is established, the question is how functional such alternatively spliced (Becker-type) dystrophin is.  Maybe what you need is at least 30% of such dystrophin to have a functional impact on DMD.  

Sustained 6 MWT stabilizations common

Supporters of eteplirsen like to point out that the proof that it works is in the apparent disease stabilization over 52 weeks or more when measured in terms of the 6-minute-walk test.  However, it appears from natural history studies of DMD that such walking stabilizations are common.  In fact, depending on the age, you would even expect an increase in walking ability over 52 weeks.  Thus, arguing that the 6 MWT data are proof for drug efficacy is like arguing that Stable Disease in an uncontrolled cancer trial is evidence that a drug works.

Confounding the 6 MWT data is the fact that it is an effort-based test and that the separation between drug and placebo was only observed after unblinding of the data and dropping out the worst performers. In the very words of Sarepta itself and clinical collaborators when discussing clinical data from the competing exon-skipping drug by Prosensa and GSK:

'In terms of the clinical efficacy, the PRO051 study claims that eight of 11 boys who were ambulant at entry to the extension study showed improvement in the 6-min walking test of 35·2 m (SD 28·7) after 12 weeks' treatment; however, this change was not significant. Moreover, several of these children were younger than 7 years and, according to longitudinal observation, boys younger than 7 years with Duchenne muscular dystrophy gain motor function. Additional confounding factors are the variability in the walking test (SD 36 m) and the powerful placebo effect of open-label studies. Despite these limitations, this observation is encouraging.'

I don’t hold out myself to be an expert in DMD, but as a skeptical scientist I believe there remain too many open questions around the eteplirsen data and trial conduct to make me feel comfortable.  Add to this the almost religious support by a strange alliance of investors, patient groups, politicians, and journalists.  Spending half a million per patient per year on a drug that may turn out to be ineffective and have side effects would neither protect patients nor help the cause of RNA Therapeutics.  If patients are so keen on the drug, and I'm all in favor of patient choice, then why not provide them with eteplirsen based on compassionate use?

Last but not least, why newly Cambridge, Mass-based Sarepta had to issue the press release on the Minutes of their FDA meetings just minutes after the Boston marathon bomb blasts, is a mystery to me.

Disclosure: no position in Sarepta.

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RNAi Therapeutics Arriving in Oncology

Yesterday, Tekmira presented data from the dose escalation portion of the phase I trial with TKM-PLK1 for the treatment of solid tumors.  I agree with the company that the fact the drug candidate was associated with clinical benefit in 4 out of 9 evaluable patients receiving 0.6mg/kg or more versus no such association at dosages below is 'highly suggestive' that TKM-PLK1 has anti-tumor activity indeed.  I also agree with their decision to pursue the neuroendocrine tumor indication for their first phase II study later this year based on the present data.

What I would like to focus on today, however, is much more exciting data contained in the TKM-PLK1 data as it relates to RNAi delivery to oncology…the key obstacle in making RNAi Therapeutics work for cancer.  Specifically, the long-circulating 2^nd gen SNALP formulation employed in TKM-PLK1 exhibited vastly improved pharmacokinetics over the short-circulating 1^st gen SNALP formulation employed in Alnylam’s ALN-VSP02.  Whether cancer in the liver or not, long circulation times are critical in order to take advantage of the Enhanced Permeability and Retention (EPR) effect that is at the heart of nanoparticle-based approaches in oncology.

It is for this fact that I am optimistic that the ‘highly suggestive’ data (which you invariably will hear about in phase I cancer studies involving a mix of late-stage cancer patients such as this one) are for real.

10-Fold More siRNA at 24 hours

Slide 13 of the AACR presentation shows that at 24 hours, around 1500 nanograms siRNA was present in 1ml of serum at the maximally tolerated dose of 0.75mg/kg.  This compares to around 150 nanograms of combined KSP and VEGF siRNA per ml at this time point for ALN-VSP02 as presented at ASCO2011.  If you’ve ever been at the bench and performed siRNA transfections in tissue culture cells, 1.5 microgram siRNA in a ml of media is overkill, and in this case it is real tumors that are served by blood containing such large siRNA concentrations- for days.

Considering the PK profile, it is not surprising that the one paired tumor biopsy obtained so far showed RNAi activity according to 5’ RACE, whereas such detection was rare in the ALN-VSP02 trial.  Clearly, more tumor biopsies have to be taken in the dose extension phase of the study (which apparently is recruiting quite well).  This will also be important for assessing siRNA concentrations in the tumors themselves.

Selecting the Right Patients

A heterogeneous patient population as in this trial alone obviously makes it very difficult to select the right cancer types for future clinical development.  There are a number of biomarker strategies that Tekmira could, and in my opinion, should employ in their future development.

Based on target biology, the KRAS mutation status should be given high priority.  KRAS-driven cancers are thought to be highly dependent on PLK1 expression.  Related to this, PLK1 expression itself could be used as a guide in patient selection.

A more unusual strategy would be to use imaging technologies based on nanoparticles which presumably rely on the same EPR effect in order to assess whether the tumors are amenable to EPR effect (alternatively, a simpler perfusion test may provide similar insights).  I could imagine that the fact that neuroendocrine tumors seem to respond to TKM-PLK1 is a result of such a favored effect.  Finally, another (admittedly less likely) selection strategy considering delivery would be to target tumors with high expression levels of LDLR-like receptors which are thought to be important in the cellular uptake of SNALP particles.

And last but not least, for those that like to think of comparative values, with a much better target and PK profile over ALN-VSP02, TKM-PLK1 may now be considered the lead RNAi Therapeutics oncology candidate. 

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