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[10B] History of Morpholinos: From pipe dream to practical products

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History of Morpholinos:

From pipe dream to practical products

James E. Summerton, Ph.D.

Email: jsummerton@gene-tools.com
Phone (541) 929-7840 ext. 1161

GENE TOOLS, LLC
1001 Summerton Way
Philomath, Oregon 97370, USA

22 April 2016

(author’s version – updated in Nov. 2016)

1.  Overview: from 1969 to 2016

Conception  

Evolution of Morpholino antisense oligos began in 1969 with my conception of a drug design strategy expected to provide treatments for most or all viral diseases, and perhaps cancers and a host of other diseases as well.  That strategy is now referred to as “antisense” because it entails developing and using an antisense sequence complementary to the sense genetic sequence one wishes to block or alter.

Research applications

The first gene blocking agents evolved through many stages to our current  Morpholino antisense oligos, which were launched commercially in the year 1999 as custom research reagents which have been used mainly as gene modulating agents for studying the intricate cascades of gene activations and deactivations which are precisely controlled with respect to both time and position in rapidly maturing embryos, particularly zebrafish embryos, a preferred model organism in the developmental biology field.  Morpholinos, micro-injected into fertilized eggs, have dominated antisense applications in the developmental biology field (1, 2, 3) because only Morpholinos:  i) provide adequate sequence specificity;  ii) are completely stable in biological systems; and,  iii) are generally free of the off-target effects which commonly plague other antisense structural types, including S-DNAs, siRNAs, and shRNAs.

Therapeutics — the low-hanging fruit  

The lack of a technology for safely delivering Morpholinos into the proper subcellular compartments in animals (including humans) has long deterred broad therapeutic applications of Morpholinos.  However, for the special case of muscular dystrophy the underlying mechanism of the disease itself facilitates delivery of therapeutic levels of Morpholinos into just those cells in need of the therapeutic treatment (muscle cells which self-permeabilize due to a lack of dystrophin).  To exploit this special case, the biotech company, Sarepta Therapeutics (previously named: ANTIVIRALS Inc., which I founded in 1980), has developed a safe and moderately effective Morpholino drug for treating muscular dystrophy, and has carried that drug through successful Phase 1, 2, and 3 clinical trials (completed in late 2015).  In September 2016 that drug was approved for clinical use by the FDA..

Therapeutics — the final component?  

A safe and efficient delivery technology has long been lacking in order for Morpholinos to fulfill their full promise of therapeutics for a broad range of diseases.  As of March 2016 it appears researchers at GENE TOOLS have finally developed a delivery technology capable of providing safe and efficient delivery of Morpholinos in vivo.  While this new delivery technology is still being optimized, preliminary results suggest that Morpholinos will now finally be able to provide the long-promised therapeutics for a broad range of difficult-to-treat diseases.  Barring unexpected problems, our first application of these new delivery-enabled Morpholinos will comprise custom cocktails for curing any patient’s cancer.     

2. A difficult start toward antisense: 1969 – 1979

A key seminar

In 1969 I was a graduate student who had recently joined the lab of Dr. Christopher Mathews in the Biochemistry Department of the new medical school at the University of Arizona in Tucson.  Dr. Mathews’ lab was focused on the biochemistry of nucleic acids.  The department’s weekly seminar was being given by   B. R. Baker, a famous scientist in the field of drug design.  His topic was “active-site-directed  irreversible-enzyme-inhibitors for viral diseases”.  His development strategy started with a known substrate for both the viral enzyme and the corresponding human enzyme.  A large number of simple chemical derivatives of that substrate were made and tested for increased binding to the viral enzyme, but not to the human enzyme.  Any derivatives which afforded greater binding to the viral enzyme, but not to the human enzyme, were carried through one or more additional cycles of derivatization and testing until several compounds were produced which had a strong preference for binding to the viral enzyme, but not to the human enzyme.  The best compounds were then derivatized with each of a wide variety of reactive groups until a compound was generated that irreversibly bound to the viral enzyme, but not to the human enzyme.  Such a compound was expected to constitute a near-final therapeutic for that virus.

In essence Baker was:   i) identifying a target viral structure;  ii) then laboriously generating a complementary structure; and finally, iii) adding a reactive group to the complementary structure such that upon binding of the complementary structure to the target viral structure the reactive group covalently linked the two structures and thereby inactivated the target viral structure.

Initial conception  

During Baker’s seminar it occurred to me that if one switched from targeting viral proteins to instead targeting viral nucleic acids, then as much as 99% of the development effort could be avoided, and one should be able to develop therapeutics for a hundred different viruses almost as easily as for 1 virus.  In this drug design strategy one strand of the viral nucleic acid would comprise the viral target.  A complementary “carrier” genetic strand would then be prepared (using a polymerase), which would serve to carry multiple crosslinking agents.  When that carrier strand paired to its complementary target strand, the crosslinking agents would irreversibly inactivate the target strand.  The principal challenge would be to develop the crosslinking agent.  As a graduate student focused on the biochemistry of nucleic acids, it appeared that designing a suitable crosslinking agent should be simple because even in 1969 the structures of duplex nucleic acids were known at subatomic resolution, and the relative reactivities of the various sites on both RNA and DNA were well known.  A particular advantage of such a new drug design strategy was that once the required crosslinking agent was in hand, the strategy could be quickly and easily used for selectively targeting virtually any single-stranded genetic sequence – affording therapeutics for most or all viruses and virally-mediated cancers, plus a host of other diseases. 

An indelicate suggestion   

After Baker’s seminar I went up to him and enthusiastically made the rather indelicate suggestion that he should abandon his current strategy for targeting viral proteins and instead switch to a far more efficient strategy of targeting viral genetic sequences, and I laid out what I envisioned as the compelling advantages such a switch would provide, and explained why designing the key crosslinking agent should be easy in light of the precise structural information and chemical reactivities then known about nucleic acids.

My brash suggestions were not well received by the world-famous Baker.  Perhaps my red beard, shorts, and sandals, and my excessive enthusiasm for and confidence in an idea which was only minutes old, may have influenced his reception of my new drug design strategy.  While I am sure that my brash suggestions to Baker were hugely embarrassing to Dr. Mathews (Chris), my dissertation advisor who was standing beside Baker, nonetheless, even then, and throughout the nearly 5 decades since, Chris has been very supportive and helpful in my efforts to develop this genetic blocking strategy.

While that initial concept probably sounded wildly speculative, it should be appreciated that each month my company, GENE TOOLS, currently designs, synthesizes, and ships to labs around the world Morpholino antisense oligos targeted against about 500 to 1,000 different RNA sequences specified by our research customers, and these Morpholinos, which have revolutionized the developmental biology field over the past 15 years, function by the same mechanism underlying that 1969 drug design strategy.

But as will be described later herein, there has been much evolution of that initial idea.  The crosslinking agents are no longer used.  And the antisense “carrier” strand has been massively redesigned relative to natural nucleic acids in order to obviate the need for crosslinking agents, and to provide a number of superior properties not possible with natural genetic material.  Such changes also simplify synthesis and reduce costs.  Also, genetic sequencing and computers now play a major role in selecting optimal targets for maximal biological activity, and achieving very high specificity for the targeted genetic sequences.  Finally, a great deal of time and effort has gone into, and continues to go into, developing technologies for safely and efficiently delivering such compounds into the proper subcellular compartments in cultured cells and animals (including humans).

“Pipe-dream” publication  

On nearing completion of my unrelated doctoral dissertation work, for my postdoctoral project I decided to pursue my 1969 idea on targeting genetic sequences for therapeutic purposes.  To this end, in 1973 I wrote a paper describing that genetic targeting strategy, and submitted it to the Journal of Theoretical Biology.  It came back with a rejection notice, but no comments.  Five years later in 1978, after having carried out proof-of-principal work as a postdoc at Berkeley, I resubmitted that paper, now accompanied by a companion paper providing supporting experimental results generated while at Berkeley.  This time the paper was accepted and finally published in 1979 (4), carrying the original submission date of 1973.  And its companion paper was also published (5).  The letter of acceptance came with a kind note from the same reviewer who had rejected the earlier version submitted in 1973.  In that note the reviewer stated that the 1973 version had been rejected because it appeared to be just a pipe dream with no real chance of success.  The reviewer also complemented my perseverance and progress in pursuing that “pipe dream”.

Experimental work begins as a postdoctoral fellow at Berkeley  

In order to be able to pursue one’s own project as a postdoc it is generally necessary to obtain a postdoctoral fellowship. To start this process I applied for and was accepted to work on my proposed genetic blocking project at the Chester Beatty Research Institute in London — contingent on obtaining a postdoctoral fellowship to pay my salary and expenses.  With that acceptance in hand, I next applied to National Institutes of Health (NIH) for the requisite postdoctoral fellowship.

Early in 1973 I was thrilled to receive notification from NIH awarding the requested fellowship to work on the genetic blocking project at the Chester Beatty Research Institute.  However, just a few weeks later another notification came from NIH regretfully informing me that the fellowship was being withdrawn.  The reason was that in order to reduce government spending President Nixon had unexpectedly sequestered a significant portion of NIH’s approved budget.  That depressing news put an end to plans to go to England to begin developing my proposed sequence specific genetic blocking agents. 

A year of delay  

Suddenly at loose ends because of this devastating news, and needing some income to live on, I embarked on a collaborative project with an Electrical Engineer at the Univ. of Arizona who had a small grant from the US Dept. of Occupational Safety and Health Adm. (OSHA) to study the mechanisms underlying silicosis in miners and foundry workers.  That very-low-budget project (around $10,000) was carried out in an autoclave room with a borrowed spectrophotometer and centrifuge, plus a fair amount of my own red blood cells (drawn from my finger tip for each day’s experiments).  That project was quite successful in identifying the probable mechanism underlying silicosis, and the information generated in that project led to an important publication (6) that improved OSHA’s strategies for reducing silicosis.  Results from that project also led to our development of a simple, inexpensive, and efficient technology for selectively removing the pathogenic fraction of air-borne silica dust from foundries and mines, including even mile-wide open pit mines.

Progress resumes  

Early in 1974, as that silicosis project was nearing completion, I unexpectedly received still another notification from NIH informing me that the postdoctoral fellowship was again available for pursuing my genetic blocking agents.  The reason that fellowship had been reinstated was that by 1974 President Nixon was embroiled in his Watergate scandal — and so was not inclined to mess with NIH’s appropriated funds.  Since the position at the Chester Beatty Research Institute in England had fallen through the previous year due to Nixon, I applied to several labs in the USA which appeared to have programs somewhat relevant to my genetic blocking project.  Bringing NIH postdoctoral fellowship funds helped favor a welcome at several labs, and I selected the lab of Dr. H. Fraenkel-Conrat and B. Singer in the Department of Molecular Biology at Berkeley as being most suitable for the genetic blocking project.

On arriving in Berkeley in the Summer of 1974 it was clear that more chemical synthesis expertise and equipment would be needed than was available in the Molecular Biology Department.  Inquires led to Dr. Tinoco in the Chemistry Department, which was just across the street from the Molecular Biology Dept.  Dr. Tinoco, in turn, suggested the younger Dr. Paul Bartlett (so called because there were two Dr. Paul Bartletts in the Chemistry Dept. at that time) might be interested.  Bartlett turned out to be the perfect mentor and collaborator, providing the practicalities of organic synthesis needed for designing and synthesizing the crosslinking agent for the genetic blocking strategy.  Because my graduate training was as a nucleic acid biochemist, advice and assistance from Bartlett in organic synthesis was invaluable for the project. 

Research associate at National Jewish Hospital and Research Center  

When my two-year postdoctoral fellowship expired at Berkeley I obtained another fellowship from NIH for a research associate position for the purpose of continuing the genetic blocking project.  With that funding in hand, I lined up a position at the National Jewish Hospital and Research Center in Denver, Colorado.  Building on the work carried out at Berkeley, the focus now shifted from chemical synthesis of the crosslinking agent, to its attachment to a suitable viral genetic sequence, and then assessment of its capability to selectively crosslink and thereby inactivate the complementary viral genetic sequence.  That phase of the project went well and completed the work we (Paul Bartlett at Berkeley and myself at National Jewish) needed for a publication describing the design and implementation of this genetic blocking strategy.   The resulting paper was submitted to and published in the Journal of Molecular Biology in 1978 (7).

Assistant professor-senior research at Oregon State University (OSU)  

After 18 months at National Jewish in Denver, I was offered an assistant professor-senior research position in the Biochemistry-Biophysics Department at Oregon State Univ. (OSU) by Dr. Christopher Mathews, who had been my dissertation advisor at the University of Arizona, and who had recently been appointed Chairman of the Biochemistry-Biophysics Dept. at OSU.  On my arrival at OSU in Feb. of 1978, Chris made available one of his labs suitable for continued development of the genetic blocking agents.  Soon after arriving I began a fruitful collaboration with Dr. Dwight Weller, a professor of organic synthesis in the Chemistry Department just across a parking lot from my lab in the Biochemistry-Biophysics. Dept. at OSU. 

In early work at OSU  I found that after attaching my crosslinking agent to a carrier strand of nucleic acid, that agent slowly underwent substantial intra-molecular reaction which short circuited the genetic blocking strategy.  To avoid that problem, cross-complexing agents were designed to stabilize the carrier/target duplex much like the crosslinking agents, but without the covalent crosslink.  However, after synthesizing these cross-complexing agents, they were also found to suffer serious limitations due to intra-molecular interactions that blocked pairing to their target genetic sequences. 

These limitations provided the motivation in 1979 to devise a radically different design strategy for blocking specific gene sequences.  The idea was to design a set of 4 subunits, each of which could hydrogen bond to three polar sites facing the major groove of one of the four oriented base pairs in duplex DNA (those being: A:T, T:A, G:C, and C:G).  By linking these four subunits in a selected order, the resultant oligomer was predicted to bind and block the functioning of any selected gene.  Such oligos were named “anti-genes”, because unlike antisense agents, which block the sense RNA transcripts, anti-genes were designed to block specific genes in their duplex-DNA form.  Another version, with a different backbone structure, was designed to block A-form DNA/RNA and RNA/RNA duplexes.

1978 – 1979:  Birth of the antisense field  

The antisense field is considered by many to have started in 1978 with two publications by Paul Zamecnik and Mary Stephenson at Harvard.  They reported the use of a 13-mer antisense DNA oligo (purchased from a commercial source) to inhibit translation of the sense Rous sarcoma viral RNA in a cell-free translation system (8).  In a companion paper they also reported results from experiments in cultured cells wherein that same 13-mer antisense DNA was added at the same time as an infecting Rous sarcoma virus – resulting in inhibition of virus production (9).  Those two papers played a major role in bringing the antisense therapeutics strategy to the attention of researchers studying gene function, and others interested in new approaches to drug design.  Being published in a high-impact journal (Proc. Natl. Acad. of Sciences), coming out of Harvard, including results from a live virus in cultured cells, and utilizing an antisense oligo that could be purchased from a commercial source, all contributed to a very high impact for those two papers.

Also in 1978 Paul Bartlett and I published an extensive antisense paper in the Journal of Molecular Biology which demonstrated the functioning of our sequence-specific crosslinking agent for specifically linking a viral RNA to its complementary DNA (7).

Also in 1978 the first patent was issued in the antisense field (US Patent 4,123,610).  This was issued to me and Paul Bartlett and assigned to National Institutes of Health.

The following year (1979) two of my antisense papers were published in the Journal of Theoretical Biology.  The first paper, which described my basic antisense therapeutics strategy, was initially submitted in 1973, but rejected, and then a more extensive version was re-submitted in 1978 and published in 1979 (4).  A companion paper focused on design of the crosslinking agent used in that genetic blocking process (5).

Also in 1979, Paul Miller, Paul Ts’o, and coworkers at Johns Hopkins University published a paper on a new uncharged linkage structure for DNA (10).  While that methylphosphonate linkage was not usefully incorporated into an antisense oligo until the mid-1980s (11, 12), it, and an earlier phosphotriester linkage type developed by the same group (13), suggested an effective means for achieving good stability of antisense oligos in biological systems.  This was significant because achieving good stability in biological systems is a major challenge in developing antisense agents.

From that modest beginning in 1978 and 1979, between the mid-1980s through the end of the 1990s the antisense field ballooned into a huge research and development effort which led to dozens of different antisense structural types, involved many hundreds of researchers, was funded by hundreds of millions of dollars from NIH and other research funding agencies, and was pursued by most of the major pharmaceutical companies.

1980 – Leaving OSU and starting the first antisense company  

My newly-conceived anti-genes were an exciting conceptual breakthrough (detailed years later in US Patent 5,166,315) and appeared to have great promise for therapeutics.  Therefore, before moving forward with their reduction to practice I started looking into the terms of my grants and other sources of funding to make sure there would be no impediments to taking anti-genes all the way to therapeutic products that could benefit humanity.  In the course of looking through the fine print of my grant from the US National Institutes of Health I was appalled to find a clause which specified that for grants to non-profit and governmental entities (such as OSU), for any patents issued on inventions made with those grant funds, said patents could not be licensed exclusively for more than 5 years to any non-governmental organization, such as a major drug company.  The consequences of that clause was that if my new anti-gene design strategy worked out as hoped, then it would be virtually impossible to ever get any of the resulting anti-gene therapeutic products to patients.  This is because if no patent application was filed then no drug company would be willing to put up the hundreds of millions of dollars required for development and regulatory approvals of each of the therapeutic products — because any such company would lack market exclusivity which is essential in order to recoup the huge development and regulatory expenses required for new drugs.  If instead a patent application was filed and a patent issued, again no drug company would be willing to fund the development and regulatory approvals because their maximum of 5 years of license exclusivity under that patent would have expired long before they could ever get any of the products into the market.  Further, the US Government, and virtually all other governments, do not fund late-stage clinical development and regulatory approvals – they always leave that extremely expensive component of drug development to be paid for by commercial entities.  Thus, I concluded that continuing on at OSU with funding from government grants would be a complete waste of my time, and a waste of government funds, because even if the technology was successful, no therapeutic products were likely to make it to patients.

In sharp contrast, at that time if a for-profit company was awarded a grant from NIH, that absurd clause (limiting license exclusivity to no more than 5 years) would not apply.

At that point it appeared I had two choices:  1) I could continue working at OSU on my newly conceived anti-genes, but with the likelihood that such anti-genes would never be used as therapeutics for human patients; or,  2) I could abandon my position and lab at OSU, give up my funding from NIH (and several other sources), and take the risk of starting up a private company to develop my genetic blocking agents.

The folly of developing a therapeutics technology that could never serve patients was unacceptable.  This, and other factors, motivated me to start  ANTIVIRALS Inc.(AVI) on 1 January 1980, the first company focused on developing antisense therapeutics.  Note that in 1997 my current company, GENE TOOLS, LLC, was spun off from  AVI  to focus on custom Morpholinos for the research community.  In 2002 ANTIVIRALS Inc. was renamed “AVI BioPharma Inc.”, and in 2012 again renamed “Sarepta Therapeutics”.

Key contributions to developing Morpholino antisense oligos  

Founding a new biotechnology company was a huge risk, and without key contributions from my wife it would not have been feasible.  Her contributions were as follows.  In 1977 while working at National Jewish in Denver, I met a young teacher, Patricia Rusnak (Pat), on a hike up a 13,000-foot-high mountain near Denver, and not long thereafter we became engaged.  Late in 1977  I received an offer of a research faculty position at Oregon State University in Corvallis, and in Jan. of 1978 Pat and I married and a week later we packed up and moved to the beautiful city of Corvallis, Oregon.  Since my position at OSU was not tenure track, and since I was pursuing a then-quite-speculative line of research, Pat prudently elected to enroll at the local community college to add degrees in computer programming and in accounting to her Masters in Education.  Her foresight in getting that additional practical training would soon make a major contribution to the future development of Morpholino antisense oligos – by providing us with an income while I focused on starting up my new 1-person company.  Further, during her years of teaching Pat had frugally saved a significant portion of her income — which allowed her to make the down payment on our first home in Corvallis.  That home was to also play a key role in the future development of Morpholinos.  Specifically, the basement served as my company’s first very-low-cost laboratory.  In addition, several years later that basement laboratory served as the location for a site visit from NIH.  The site visit team was there to judge whether or not my proposal for a Small Business Innovation Research grant could likely be successful in my rather unconventional lab facilities.  Happily, they concluded the project had a reasonable chance of success and so the grant was approved.  But approval was contingent on moving the lab from the basement of our house (by then with three small children living upstairs) to a new facility, and NIH added $ 15,000 to the requested grant in order to cover the cost of the move and the increased operating expenses.  Accordingly, soon thereafter I set up a small well-vented organic synthesis lab in a 400 square foot abandoned tombstone dealership. 

3.   Antisense comes of age

Development of simple prototype anti-gene   

Research in 1980 was spent designing, synthesizing, and testing a very simple prototype anti-gene in order to test the feasibility of gene blocking by targeting major-groove-binding sites in duplex DNA.  While results appeared promising, it also became clear that developing effective anti-genes for binding specific sequences of base-pairs in duplex DNA would likely require much time and resources – far more than my fledgling 1-person company could muster.

Return to antisense  

Therefore, in 1981 I redirected my focus to antisense agents targetable against single-stranded RNA sequences.  In my renewed focus on antisense agents my designs were strongly influenced by the Miller and Ts’o strategy of utilizing a non-ionic backbone to prevent degradation in biological systems (10).  I also postulated that a properly designed antisense oligo with a suitable non-ionic backbone might bind its target sense strand with sufficient affinity that there would be no need for a cross-linking agent — thereby avoiding the intra-molecular troubles I’d previously encountered with antisense sequences carrying cross-linking and cross-complexing agents. 

By the mid-1980s: the antisense drug design strategy was rapidly gaining popularity; ANTIVIRALS was no longer just a 1-person operation; and, we were making very good progress toward increasingly effective antisense structural types.  As a consequence, it was becoming easier to raise funds for our research.  This allowed several moves to progressively larger facilities, and hiring an increasing number of researchers.

During the 1980s at ANTIVIRALS Inc. we developed and tested a substantial number of antisense structural types.  Of those many structural types, Figure 1 shows the three most significant types in our long progression of structures with increasingly desirable properties.  It should be noted that between structure A and structure B there were about 6 other structures made and tested, but judged inadequate for our purposes.  And between structure B and structure C there were about a dozen structural variations made and tested, but again judged less than optimal for our purposes.

Antivirals' antisense structural types

Finally, structure C in Figure 1 (our current preferred structure) was found to have all the properties we had long been searching for.  It was first synthesized and tested in 1989.

By the year 2000 antisense oligos of the structure C-type were fully developed and had been carefully tested at the biophysical and biochemical levels, and at the cell culture level, and testing had begun in living animals.  Extensive experimental evidence at all of these experimental levels clearly indicated that our Morpholino structural type (structure C of Figure 1) had an unmatched combination of properties which, in complex biological systems, led to their greatly out performing all of the popular competing structural types, particularly in regard to the key properties of: sequence specificity; general absence of off-target effects; long-term stability; and predictable targeting (17, 18, 19, 20, 21, 22).

Aside from the new structural types being devised and developed at ANTIVIRALS Inc., during the 1980s and into the 1990s most new antisense structural types were being developed at universities and national laboratories (NIH and the FDA).

Figure 2 shows the most popular of those competing antisense structural types reported by other groups in the years between 1978 and 2001.

Popular antisense structural types

Table 1 lists the most used antisense structural types, with a qualitative comparison of the key properties required for effective antisense activity (17, 18, 19, 20, 21, 22).

Qualitative comparison of properties of antisense structural types

While ANTIVIRALS Inc. was the sole antisense company from 1980 until the late 1980s, beginning in 1987 four more antisense companies and one anti-gene company were founded.  These were:  Gilead Sciences Inc. in 1987;  Genta Inc. in 1988;  Hybridon Inc. in 1989; ISIS Pharmaceuticals Inc. in 1989; and the anti-gene company, Triplex Pharmaceutical Corp. in 1989.

While ANTIVIRALS Inc. devised and developed multiple new antisense structural types, in sharp contrast, the principal business strategy of the newer companies founded in the late 1980s (and generally funded by venture capital) was primarily to use established antisense structural types to target particular genetic sequences associated with known diseases, and then take the resultant antisense oligos into clinical trials.  Because of the multiple serious limitations of structural types D, E, F, G, and H (see Figure 2 and Table 1), and virtually all of the other less popular structural types, the business strategy of taking antisense oligos of those inadequate structural types into clinical trials has been pretty much a disaster — leading to many failures to obtain regulatory approvals, wastage of multiple billions of dollars, and loss of creditability of the antisense drug design strategy.  And even in those rare cases where FDA approval was obtained (eg., Vitravene, approved in 1998), at best the antisense products are of only marginal value to patients – generally because of the inadequacy of the respective structural type.

Not surprisingly, the big pharmaceutical companies have almost all abandoned the antisense therapeutics strategy — with the exception of structure H of Figure 2 (siRNA and its precursor, shRNA), which is currently the latest fad in the antisense field.  However, I believe the popularity of this structural type is destined to soon fade when researchers begin to do rigorous sequence specificity studies.  This is because of a serious limitation of siRNAs – that being there are potentially hundreds of RNA targets for each siRNA (22).  This is a consequence of the seed sequence, which determines the sequence specificity of the siRNA, recognizing too little sequence information to assure an adequate level of specificity for the targeted RNAs. 

I contend that for antisense drugs to truly provide substantial benefits to patients, the drug developer needs to:

  1. develop an antisense structural type (such as the Morpholino type) which provides all of the key properties listed in Table 1;
  2. develop a safe, effective, efficient in vivo delivery system sufficient to get adequate amounts of the antisense drug into the cytosol of cells of the tissues to be treated; and,
  3. only after steps1 and 2 are achieved should clinical trials proceed on a specific drug.

The final frontier  

I believe that the final frontier in the antisense field entails achieving safe and efficient delivery of antisense oligos into the cytosol/nuclear compartment of a broad range of cell types in living animals, particularly humans. 

I have been working toward this delivery objective since 1993, and it appears that the crucial breakthrough was finally achieved in March of 2016 (last month).

If this March 2016 advance in delivery technology continues to be as promising as it now appears, then I suspect the long-promised flood of antisense therapeutics may finally arrive, perhaps beginning as soon as 2017. 

In view of this new delivery advance, our first objective is to develop custom cocktails of Morpholinos for curing any patient’s cancer (31, 32).  Each component of the cocktail for a given patient will be targeted against one of the RNA transcripts absent from the patient’s normal cells, but found (by sequencing a biopsy sample of the patient’s cancer) to be present in and essential for the viability of that patient’s cancer.  Such cocktails are explicitly designed to destroy the cancer without harming the patient.

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