Genetic Medicines

I attended a conference entitled ‘Personalized Medicine: Concepts, Promises, Successes and Challenges,‘ organized by the Jacques Loeb Center for the History and Philosophy of the Life Sciences and the National Inst. for Biotechnology in the Negev (NIBN) at the Ben Gurion University in Beer Sheva, where I am a Visiting Professor of Chemistry.  What I was disappointed to discover was that most of the speakers pointed out the difficulties and problems, both medical and ethical, of ever reaching a truly successful personalized approach to medicine,

This is especially true of such diseases as cancer that are now known to be genetically complex, and in which a series of genes are turned on and turned off compared to the “normal,” so that it is virtually impossible to overcome this genetic heterogeneity.  A great deal of work is being done to characterize this heterogeneity, but unfortunately there has been little success in overcoming this problem.  One of the reasons for this is  that the medicines being developed against cancer and other diseases are small molecules that selectively inhibit specific proteins that are produced in aberrantly functioning cells such as metastatic cells.   It is impossible to achieve any level of inhibition of a series of such proteins with small molecule drugs that are not selective enough.

If I say I have a possible solution to this problem, very few people will believe me, but the fact is that cancer and like disorders are genetic diseases, that involve the malfunction of at least several genes (due to serial mutations).  In order to cure a genetic disease I maintain that it is necessary to use a genetic approach to therapy, in other words to develop genetic medicines.  I used this title for an article I published in Scientific American in 1994 (Cohen, JS, and Hogan, M. The new genetic medicines, Scientific American271 (6): 50-55, 1994.).

What are genetic medicines? They are molecules that have a base sequence that can bind selectively to a specific target sequence in the messenger RNA of a gene that is carcinogenic, that is causing aberrant cell behavior, i.e. cancer.  Note that it is rarely possible to bind a complementary sequence to DNA, that is covered in proteins and carefully protected, since it contains the genetic information.  But, as originally predicted by Francois Jacob and Jacques Monod, and subsequently proven by Jacob, Brenner and Meselson in 1961 at Cal. Tech., there are messenger molecules that are RNA (mRNA) that take the sequence of DNA (called the sense sequence) from the gene out into the cell, where they are expressed as protein via the protein biosynthesis mechanism involving the ribosome.  Naturally, the complementary sequence is called the antisense sequence, and this is the name give to the approach of using chemically modified analogs of DNA as drugs to inhibit gene expression via the interaction with mRNA.

Now it is true that this antisense approach has serious problems, for example, (1) Stability. It is necessary to design and synthesize chemically modified analogs of DNA to ensure that they are stable in the cell, protected from the prevalent enzymes that degrade foreign DNA, known as nucleases.  For example, we pioneered the use of analogs with a single substitution of a sulfur atom in place of an oxygen on the phosphate group of DNA (eight such PS (thio-phosphate) analogs have been approved by the FDA for human use); (2)  Uptake. It us very difficult to get such large molecules into the cell where they are required to function.  But this can be overcome by designing the analogs to be more hydrophobic than normal DNA, or using liposomes to deliver them to the cells (we carried out studies on liposome delivery of antisense oligos, in collaboration with Yehezkel Barenholz at Hebrew University, some years ago); (3) Cost. The cost of such specialized molecules is high, but if they work they would save even more money from futile treatments with small molecule drugs that eventually fail; (4) Biological therapy. There is a trend to use biological approaches to therapy, but these tend to be complex and not easily controllable, so that approaches using gene therapy and what is known as siRNA, naturally occurring gene silencing RNA’s, have not so far been successful; (5)  The molecules are complex and in some cases form sequence dependent structures that reduce their efficacy.

However, my contention is that we must work hard to overcome these difficulties in order to take advantage of the truly great potential of genetic medicines.  This approach  has the potential to overcome the very genetic heterogeneity oi cancer that has prevented current progress by using a  cocktail of DNA analogs with selected base sequences that could be the basis for a truly personalized therapy.

 

 

 

Advertisements

A New Non-natural Genetic Code

I attended a fascinating lecture at Ben Gurion Univ. given by Dr. Floyd Romesberg of the Scripps Institute, La Jolla, California, entitled “A semi-synthetic organism that stores and retrieves increased genetic information.”   This title my be somewhat obscure to people, but it comprises a remarkable breakthrough.

All organisms on earth function through the information contained in the base sequence of DNA.  This involves four base letters (A, T, G, C) that form two base pairs (A=T, G=C).  This is the universal genetic code that has evolved thru millennia. But, there are in fact many more such bases that exist with chemical modifications that are not found naturally in DNA.  Prof. Romesberg and his collaborators decided to set out to see if they could isolate or design another non-natural base pair (note I use the word non-natural rather than unnatural, which has connotations of being weird).

In doing so they did not start with preconceived ideas, but decided to test 1,800 such non-natural bases to see if they could be involved in base pairing.  This involved a great deal of work and many false starts.  But, after working for 20 years with many post-doctoral fellows, they did in fact isolate a base pair that mimics the role of the two natural base pairs.  Two points about this base pair (let’s call it X=Y), first the bases found by the trial and error selection were very hydrophobic (that is they prefer to avoid being in water solution and tend to fit well into the water-free interior of DNA.   Second, although molecular structures of short pieces of DNA (oligonucleotides) containing the non-natural base pair have been determined, the two bases (X and Y) are not actually paired with hydrogen bonds as are the canonical base pairs (A=T, G=C), but they nevertheless appear to form such hydrogen bonds when involved in biochemical processes.

Romesberg and his associates were able to actually integrate this non-natural base pair X=Y into the genome of a microorganism, E. coli, that functions very well and produced proteins that have a non -natural amino acid derived from the non-natural base pair by the natural genetic process.  What does this mean?  It means that chemically modified proteins can be made in situ from micro-organisms that might have significant biological/therapeutic properties.

But, on another level it shows that man’s ingenuity is able to manipulate the genetic code, indicating that it is not unique or of divine origin. But, before people start worrying about unnatural organisms existing in the world, I point out that it took 20 years and testing thousands of non-natural DNA bases to arrive at one novel non-natural base pair that works in the biological world.  So no need to worry about strange mutated organisms just yet.

Another Biological Defense System

Following on from my previous blog article about how do polyaromatic hydrocarbons (PHC’s) like benzo-a-pyrene cause cancer?  It took many years of research by many people to come up with the answer to this question.  Ironically it turns out to be due to a little known biological defense mechanism in our systems that works too efficiently.

Everyone knows about the immune system, that is a complex biological mechanism that recognizes foreign substances in our system and removes them.  This includes bacteria and many chemical substances.  The immune system is so flexible that it can even recognize previously unknown chemicals and remove them from the body.

But, there is a catch, the immune system cannot deal with very hydrophobic substances that are essentially water-insoluble, such a PHC’s.  To remove them from the system (even in very small quantities) another defensive system was developed called the cytochrome P450 system.   This is parallel to but not as widely known as the immune system.  A cytochrome is a protein that is involved in oxygen transfer and cytochrome P450 has a very unusual peak in the UV/vis spectrum, at 450 cm-1 that identifies it.  What this cytochrome does is hydroxylate the aromatic molecule, i.e. it adds an oxygen atom to them that becomes a hydroxyl group to make them soluble and therefore excretable.

But when there is a certain structure of PHC such as benzo-a-pyrene, this system sometimes results in a mistake and in a few percent of cases an epoxide is formed, which contains a very chemically active oxygen.  These planar hydrocarbons being hydrophobic also bind avidly to DNA, they intercalate (insert) between the stacked base pairs.  In doing so, if they are epoxides, they react chemically with the DNA and this leads to mutations and cancer because the DNA cannot be read by the required enzymes.

So this is how chimney sweeps got a special kind of cancer, the PHC’s in the soot, that got into their blood system were expoxylated by the cytochrome P450 system and reacted with the DNA to cause mutations.  Other substances react in a  similar way, for example, the most toxic/mutagenic substance known is aflatoxin.  It is found in the fungus that grows on the inside of the shells of peanuts, and forms a white shiny surface.  This fungus grows very well when the peanuts are stored for long periods in damp conditions.  Nowadays, the storage conditions are kept dry and the amount of aflatoxin is tested and regulated by the FDA in all peanut products, including peanut butter.


Clarification: Please note that there are two distinct uses of the term “aromatic”; in the ordinary meaning to describe a smell such as a perfume, or to describe a series of chemical compounds based on benzene, these two meanings are not synonymous.  The latter compounds can smell and can be highly colored, but not necessarily, and they are not all carcinogenic.

 

What are Aromatic Compounds?

At the same dinner that I mentioned in my previous blog article on Einstein’s beliefs, I was quizzed on the nature of carcinogenic substances and how the body handles them.  I have found that most lay people are profoundly ignorant of basic chemical and biochemical concepts.  I was asked what are “aromatic” compounds and are they carcinogenic because they are volatile and because of their smell?

It is interesting that the first example of cancer discovered in the early 1800’s was that of chimney sweeps and especially their young helpers, who actually went into the chimneys to clean the soot and carried sacks of soot on their back.  They developed growths that were later diagnosed as cancers, that clearly resulted from contact with soot.  But, how and why?

Actually “aromatic” describes a whole class of organic compounds (i.e. compounds made up of carbon) that are based on benzene.  Starting in the early 19th century, carbon compounds were characterized in which carbon has four valencies, i.e. makes four bonds with other atoms, such as hydrogen.  Thus the simplest such compound (known as hydrocarbons) is methane CH4 (known as natural gas).  Derivatives of these are the well-known alcohols, such as methanol CH3-OH and ethanol C2H5-OH.  But, then the stable substance benzene was discovered to have the composition C6H6.  No one could then figure out what its structure might be.

The problem was solved by a German chemist named Kekule who related that in 1858 he fell asleep on the upper deck of a London bus, and dreamed of a snake swallowing its tail.  When he awoke he realized that benzene must have a cyclic structure, in fact a six-membered carbon ring with one hydrogen attached to each carbon.   It was subsequently discovered that there are many aromatic compounds with benzene rings fused to each other (called poly-aromatic hydrocarbons).  Soot is an excellent source of such compounds, and it was eventually shown that benzo-a-pyrene is the active ingredient in soot that is highly carcinogenic and mutagenic and causes cancer.  The next question is why?

Did Einstein Believe in God?

I was invited to a dinner and at the dinner I met an interesting man named Bernard who was a physicist although he was religious.  When he found out that I am a biochemist he asked me several direct questions, and we had an interesting conversation about metabolism, drugs and cancer.  Then he also asked me if I believe in God.  I said that I did not (even though I was also wearing a kippa as a courtesy).  He asked me why and I explained this by saying that I did not have a religious upbringing and that I am a scientist.  Then Bernard made the statement that “Einstein was certainly a scientist and he believed in God.”

At that point I had to correct him, having read Walter Isaacson’s biography of Einstein and other material. I pointed out that Einstein’s famous statement “God doesn’t play dice with the universe,” was not a statement of belief by Einstein, but rather a refutation of quantum theory, that is based on probability, that Einstein spent the latter half of his life trying to disprove (unsuccessfully).  Bernard challenged me to provide another quote that proved Einstein was not a believer in God.  I told him that I could not remember such a quote verbatim, but I would gladly send one to him.

Instead of going to Isaacson’s book, I looked up “Einstein’s religious beliefs” in Google, and indeed there was a long article there.  I chose more or less at random a short paragraph with some quotes from Einstein, and sent them to Bernard, with  a covering note saying that I agreed with him.  Here is the quote :

Einstein expressed his skepticism regarding the existence of an anthropomorphic God, such as the God of Abrahamic religions, often describing this view as “naïve” and “childlike“.   In a 1947 letter he stated, “It seems to me that the idea of a personal God is an anthropological concept which I cannot take seriously.” In a letter to Beatrice Frohlich on 17 December 1952, Einstein stated, “The idea of a personal God is quite alien to me and seems even naïve.”

This proved my point that Einstein rejected the so-called Abrahamic God and any kind of personal God, and although he remained a skeptic, he was in awe of the universe and retained some spiritual sense of a creator.

I received a pleasant response from Bernard, and a quote that he considered showed that Einstein’s was not an atheist:

Einstein stated, “I am not an Atheist.”   According to Prince Hubertus, Einstein said, “In view of such harmony in the cosmos which I, with my limited human mind, am able to recognize, there are yet people who say there is no God. But what really makes me angry is that they quote me for the support of such views.” 

My views are more atheist than Einstein.  I do have spiritual feelings when I see the amazing complexity of the animals in the world, but I know them to have been created by evolution by natural selection.  I am amazed at the complexity of molecular biology, but I see no reason to bring God into consideration to explain the interaction of bio-molecules.  I also cannot accept that the God that Jews have beseeched to protect them actually exists after the Shoah.

Science has refuted many religious views, such as that the earth is flat, that the sun revolves around the earth, that God made man from dust, that God created all the animal species as we find them, that God dictated the Torah (Bible) to Moses (writing on parchment did not appear until at least 300 years after the estimated date of the Exodus) and so on.  It is clear that religion is not equipped to handle the physical world, on the other hand science is not really equipped to handle the spiritual world.  Science deals in measurable quantities, such as length and time.  But religion deals in immeasurable quantities, like love and faith.  It is better that we keep that separation, equivalent to the separation of Church and State.

Note: I have expounded further on my views in an essay “The Tolerant Atheist” that I published in a book entitled “Of Gods and Lemurs,” available on Amazon.

.

Science and Race

There was an interesting conference, at the Jacques Loeb Centre for the history and philosophy of the life sciences at Ben Gurion University, entitled “Science, Race, Ethnicity and Identity.”  I was only able to attend the morning session  consisting of four presentations, but I found them very stimulating.

According to most popular beliefs, humans are made up of several races, such as white European, black African, yellow Asian and native Americans.  But, these are not really races at all, since each can co-habit with the other and produce offspring.  The definition of a race or species according to science is that they cannot reproduce.  Humans have been on the earth about 500,000 years at most, probably about 350,000 years.  That in scientific terms is simply not enough time for separate races to develop.

The racial ideas developed by the Nazis, that they needed to keep their “Aryan” race pure is nonsense, since there is no such thing genetically as a pure race.  All the major groups of humans in the world, that have been largely geographically separated, are genetically over-lapping.  There is diversity, but not along so-called racial lines.  The homogeneity is greater than the diversity.  This was the conclusion of Prof. Diethard Tautz, from the Max Planck Inst. for Evolutionary Biology, Germany.  According to genetic analysis, there is only one species of human being (Homo sapiens) and all others are now extinct, from genetic analysis the Neanderthals can be considered a sub-species.

Prof. Giovanni Destro-Bisol from Sapienza Univ., Italy, also said that the concept of “race’ is not useful for biologists and geneticists. But, whether we like it or not, the term “race” is useful and has entered the legal sphere.  For example, it is stated in law that one cannot discriminate on the basis of “race, religion, ethnic group, etc.”   There are organizations that seek to remove all mention of race from the law, yet the preponderance of opinion is that it is a useful concept that has meaning for most people.

Michael Gilead, a psychologist from Ben Gurion University, concluded from human experimentation, that the basis of so-called racial distinction comes from the human need to identify those who are of the same group or clan, and therefore safe,  from the others, who are not.  This is a very basic, deep inherited human response and in itself is not necessarily discriminatory.  It is only when it becomes political, that issues of rights, ownership and superiority enter into the issue.

Later presentations covered the issues of Jewish concepts of race and the genetic definitions of ethnic origins.

Fraud in Science III

The one case that looms large in the issue of fraud in science was that of the controversy over the discovery of human immunodeficiency virus (HIV), the causative agent of AIDS. In 1984, the two co-discoverers of HIV, Luc Montagnier of the Pasteur Inst., Paris, and Robert Gallo of the US Natl. Cancer Inst. (NCI), published papers side-by-side on their work.  But, subsequently a controversy erupted between them, because the HIV genome was found to be very variable, but the two sequences published by them were almost identical.  Montagnier accused Gallo of stealing a sample of his material.

A reporter for the Chicago newspaper wrote an article on the subject and Congressman John Dingell, who was Chairman of the Science Sub-Comm. of Congress, read the article and decided to hold hearings on the subject.  As a result there were a series of detailed investigations of Gallo’s lab by the FBI, that took 5 years!  It was discovered that Gallo had isolated his specimen of HIV from a sample of another virus given to him by Montagnier.  Although his scientific credentials remained intact, and the US and French Governments agreed to divide the royalties on their patents on HIV, nevertheless priority for the discovery of HIV was given to Montagnier, and he alone won the Nobel Prize in 2008.  However, several of Gallo’s co-workers were also supposedly found to have committed fraud and misappropriated funds.

Mika Popovic was a leading researcher in Bob Gallo’s Lab. He was found guilty by the Office of Scientific Integrity (OSI) at NIH of scientific fraud related to a paper published in 1984.  On that basis he was fired from the NIH and lost his pension.  But, he maintained that he was never given any right of appeal or due process and he sued NIH.  In response the Dept of Health and Human Services established an independent panel of lawyers to investigate the procedures used by OSI and they concluded it was totally faulty, relying on hearsay and not allowing any response by the accused.  As a result, Popovic was exonerated and the case against Gallo was dropped after 5 years of investigation.

Another controversial area is climate change.  Climate change and global warming has been generally accepted as scientifically based.  But, there are many cases of falsification of data and analysis in order to “prove” the urgency of the case.  The evidence for the shrinkage of many glaciers in the Alps was shown to be falsified.  The UN inter-governmental panel on climate change claimed that the Himalayan glaciers were receding, but this turned out to be fraudulent.  Temp warming date from NOAA has been found to have been serially readjusted.  Such fraud does not disprove global warming, but certainly raises doubts.

A study by a University Committee tasked with estimating the amount of fraud in science came to the conclusion in 2017 that ca. 3% of published papers are unreliable.  Science itself is not at fault – because of the need to verify and check scientific claims the scientific method is self-regulating.  But, since science is a human endeavor that can give fame and fortune there is always a human failing to fake results.