ORPHAN DRUGS & GENE THERAPY

by stevefredman

Each year 100 American babies are born with Pompe’s disease.  The children are floppy, their muscles barely work, and their heart is enlarged.  Few survive infancy.   A genetic, recessive condition, the disease is only seen when both parents carry the defective gene.

The malady is the result of an enzyme deficiency.  The kids’ cells don’t make enough lysosomal acid alpha-glucosidase, a protein that’s used to convert stored glycogen into glucose—energy.

We eat carbohydrates and sugar, and we turn what we don’t use into a storage polysaccharide called glycogen.  We stockpile the excess fuel in our muscles and liver.   Between meals, when we need sugar to keep going, we use enzymes like lysosomal acid alpha glucosidase to turn glycogen back into glucose.  Babies who lack the enzyme won’t stay alive long.

The condition was “characterized” by and named for a Dutch pathologist–Joannes Pompe.  A member of the Dutch resistance, he was executed by the Nazis in 1945.   The absent enzyme, Lysozyme, was isolated in Belgium in 1955, and the responsible GAA gene was identified in 1979.  (It differs a little from one family to another.)

The needed enzyme was first made at Duke University by a dedicated team of researchers.  Their leader, Dr. Chen, the chief pediatrician, started his quest after he went to the funeral of an infant who died of the disease.  The pastor said God must have given the child life for some reason.  Chen took the message to heart, and decided to assemble a team of Duke University researchers and go to work.

The inspiration came at the right time.  Researchers knew how isolate the gene (the segment of DNA) that was needed. Bioengineers could make copies of the gene—clone it, and plant the DNA segment into a plasmid. Plasmids occur naturally in some bacteria. They are small circular segments of DNA that replicate but are not part of the nucleus. After the new gene becomes part of its structure, the plasmid is inserted into a cell, and the cell starts making the desired protein.

After scientists spend years isolating and characterizing a gene and are ready to move on, most want to preserve and immortalize the fruits of their endeavor. So they send a copy of their gene in a plasmid to Addgene, a Massachusetts based non-profit. Since 2004, Addgene has collected and stored more than 80,000 plasmids that contain genes. The plasmids came from the labs of 4000 investigators around the world. 

The Duke researchers could have, presumably, purchased the plasmid that contained the gene that’s defective in Pompe’s disease.  They made the missing protein by inserting the plasmids into cells derived from the ovaries of Chinese Hamsters.  It’s one of the mammalian cell lines that are currently used mass production of therapeutic proteins1.  It took the researchers three years to make enough Lysozyme for their early tests.  The produced enzyme was injected into a quail that had been bred to be Lysozyme deficient.   The poor bird was in bad shape.  It couldn’t get off its back, much less fly.  Post injection the creature stood and even flew a little.

After 6 years of successful research, the Duke scientists got some manufacturing help.  Production rights were licensed to Synpac, a British/Taiwanese company with a presence in Durham, Dukes home.  Synpac, in turn “used experienced contractors to manufacture the enzyme.”  (Having done the heavy lifting, the Duke scientists gave a lot away, but they retained some royalty rights.)

Once the company had produced enough enzyme, physicians at Duke infused the protein into three kids with Pompe’s disease.  Lysozyme replacement worked.

In 2006 Synpac made a deal with Genzyme.  It was a “15 year royalty sharing agreement that was potentially worth $821 million.”  At the time Genzyme was huge.  Based in Boston, their 2010 revenue was $4 billion.  The company planned to spend more than $500 million dollars creating production facilities for Myozyme (their name for the enzyme).

The following year Genzyme was acquired by the French Pharmaceutical giant, Sanofi for $20 billion.  As part of the process Duke University was paid $90 million, and relinquished its royalty rights.   In 2016 Sanofi sold $800 million worth of the needed enzyme.

Now called Lumizyme, the enzyme is currently made in large sterile factories.  Babies with the disorder get an injection of the protein every two weeks.

In this country “according to Sanofi, the average annual cost of treatment is $298,000.”   If it works the first year it’s needed the second and third year.  By the time a child is 10 years old—if no one develops a less expensive generic product, the system (insurance companies and Medicaid) will have shelled out $1 to $3 million dollars per child and big Pharma will have been handsomely compensated.

For those who feel health care is too expensive:  You ain’t seen nothin’ yet. Scientists have started to effectively attack and control an increasing number of genetic diseases.  Insurers are not allowed to deny coverage to anyone who has a pre existing condition nor can they impose lifetime or annual coverage limits.  Given the way our economy works, the treatments and cures that create so much hope, will cost a bundle.  And no one knows how we’re going to control their price tags. 

During my early days as a Kaiser gastroenterologist a colleague, a neurologist named Frank, asked me to take over the care of a 40 year old woman with uncontrollable diarrhea and incontinence.  He had been seeing her because her legs were weak and numb and she could barely walk.  She spent her days in a wheel chair. She had a disease that affects “about 10,000 people worldwide” and is called genetic amyloidosis.  People in her family had a 50-50 chance of being born with a gene that caused their body to make a form of the protein transthyretin (TTR) that didn’t fold normally.  The substance was produced in the liver and in its normal form “carried both the thyroid hormone thyroxine and vitamin A in the blood.” People who are born with the abnormal gene were well when they were young.  By the time they reached their 30s, their bodies were saturated with the misfolded protein and it started damaging their nerves, hearts, kidneys, and intestines.  When it affected the small bowel some developed uncontrollable diarrhea. A cousin in a nearby town was a little older and acquired new problems a few weeks before B got them.  B had been sick for a few years and was badly incapacitated but she somehow accepted her fate and didn’t complain.  I couldn’t do much but I did what I could.  Following diagnosis, most people with the condition have a life expectancy of 3 to 15 years.”  

Frank (the neurologist) contacted a researcher at Boston University.  It turned out she had an unproven diagnostic tool that could predict who among children had the gene. The researcher wanted to test her tool and Frank was game.  They invited relatives from all over the country to a funded reunion.  I didn’t go, but I was told the get-together was a disaster.  Family members met distant cousins who were in different stages of deterioration.  Everyone was visibly shaken by the clear picture of what was going to happen to their bodies. 

B’s daughter C didn’t want to be tested.  She was in her late teens and she just didn’t want to know.  I don’t remember exactly when or how B died, but after she was gone I didn’t see her daughter for more than a decade.  Then one day I got a phone call.  It was C and she wanted to see me.  She had chronic diarrhea that she originally thought it was caused by colchicine, a pill she took.  When she stopped the pill the diarrhea didn’t stop and she knew what that meant. Over the years she had read everything she could about the disease.  I had too.  We both knew that in many people a liver transplant stopped the progression of the condition.  C wanted my assistance and I helped her get on the list for a liver transplant at UCSF.   The university hepatologists were initially going full tilt.  Then C said she didn’t smoke marijuana and her blood or urine test showed she had lied.  That created a problem.  There are never enough livers and some people are always dying without a transplant.  To create an aura of fairness the societies have created rules that exclude some people, like chronic alcoholics who are still drinking.   At the time marijuana wasn’t legal and a person who lied about smoking it was automatically disqualified.  I don’t remember how we convinced the transplant people to give C a second chance but we did.  They came up with a creative way around C’s infraction.  She would become a research subject.  A 32- year-old ex-convict had died in an accident.  My patient was given his liver and a 67 year old grandmother with liver cancer received my patient’s liver. In the right body C’s liver didn’t make the toxic protein.  Her liver helped the grandmother a period of time.   It was San Francisco’s first domino transplant.30

C had diarrhea and was often somewhat dehydrated. She was stable for a while, but to prevent rejecting the new liver she was on drugs that suppressed her immune system.  Then she started developing urinary tract infections and she repeatedly became septic.  The immune-suppressing drugs made it hard to treat her.  Antibiotics worked at first but the sepsis recurred.  Each time the bacteria causing the flare were resistant to the drugs.  A few years after he got the liver C became very septic and died.  During her life when I saw her she often talked about her two sons.  She wanted the boys to be tested before they considered having children, but she knew that if they had the genetic disease they wouldn’t be able to get health insurance. 

A few decades passed but I never forgot about the C and B and the disease.  Recently I read the story of a surgeon named Carlos Heras-Palou.  In 2004 he experienced pain in my hands and feet and was diagnosed with hereditary transthyretin (hATTR) amyloidosis.  As he told his story, at the time of his diagnosis Carlos was “39, married with two children aged 1 and 2, and had his dream job working as a surgeon. After my diagnosis, I could not see how we would manage as a family if I had to stop working because the disease was affecting my hands. I was worried about telling my brother and sister, and the impact of the news on them and their families. The prognosis was very bad, but after a short period of despair, I felt I had to put up a fight even if I could see no chance of winning it.”  This was before CRISPR but Carlos had read about two young researchers who, in In 2006 received the Nobel Prize for discovering interfering RNA.  Their names were Craig Mello and Andrew Fire.  In their Nobel interview they spoke of the fun of scientific research.  Mello was born in New Haven, Connecticut in 1960. His father, James Mello, was a paleontologist and his mother, Sally Mello, was an artist. The grandparents came to the U.S. from the Azores. Craig, like his father, became a paleontologist.

1986 two researchers began working together at the Carnegie institute in Baltimore.  “Craig Mello: [Laughing] we’re studying these animals, trying to figure out these really ancient mechanisms of inheritance, how its DNA information passed on, and there was something really fundamental that we didn’t understand. We figured out how to introduce DNA into the germ line so that we could get progeny that have the DNA that we added. It’s sort of like gene therapy for worms.” The worm they worked on was the size of a comma on a printed page.  It was a nematode called C. elegans and it had a nervous system, muscles and an intestine.  The creatures ate bacteria and “you can put them through hundreds of generations in a year.”  When they added the DNA the researchers also added a piece of RNA that looked like a piece of an m-RNA.  The DNA they added was supposed to cause the worm to make a protein.  The researchers, presumably could monitor the effect of the DNA by measuring the protein.  Unexpectedly, when they injected the RNA anywhere into this animal, it silenced the DNA signal.  The DNA was turned off in all the cells of the body. The researchers then developed double stranded RNA, that matched the gene, and the RNA turned off the gene. In 1998 they published the paper saying, “There’s this incredibly weird response to double-stranded RNA. You put double-stranded RNA into the animal and it will find matching information and turn it off.” That was totally unexpected. And, moreover, our paper had no explanation for it.”

In 2002 a number of prominent RNA researchers and financiers started Alnylam, a drug company in Cambridge, Massachusetts.  They were trying to use RNA interference (RNAi) to treat genetic diseases.  They began to look for a disease model, a validated gene target.  It had to be made in an organ to which a drug could be delivered, and for which gene-silencing would result in the reduction of a measurable biomarker.

In 2004, when Carlos Heras-Palou learned he had genetic amyloidosis, he visited Philip Hawkins, the clinical director at the National Amyloidosis Centre in London. They talked about a theoretical approach to fighting amyloidosis.  Perhaps they could silence the gene that encodes TTR, and block production and halt the progression of the disease.  Hawkins discussed the option with the Alnylam research team and they started working on hATTR amyloidosis.

“Within a year, the company was reliably manufacturing silencing RNAs—siRNAs. The early molecules were plagued by a surplus of negative charges, making them prone to degradation. A delivery system had to be designed and tested. To avoid immune destruction and to get the medicine to the appropriate cells scientists immersed it in very small Lipid nanoparticles.  (~100 nm in size). They administered the medication intravenously.  It took a total of 10 years to develope the medicine.

“In 2013, Carlos’ younger sister was also diagnosed with hATTR amyloidosis. She was one of 29 people who received different doses of an RNA interfering drug.”

“In the subsequent phase III trial, two-thirds of participants received Patisiran, the silencing medication, and one-third received a placebo.  Carlos was one of the 225 people enrolled in that study, which ran from November 2013 until August 2017. The results were published in July 2018 (D. Adams et al. N. Engl. J. Med. 379, 11–21; 2018) and found that the drug reduced TTR production by about 81%.. Patisiran was approved for medical use in the United States and in the European Union in August 2018. It is expected to cost around US$345,000 to US$450,000 per year.”  Alnylam is trying to create a new form of the drug that can be injected subcutaneously once every three months, instead of the current intravenous infusion every three weeks. Carlos and his sister are apparently doing well.

 (During its first 16 years Alnylam invested about US$3.5 billion into RNAi therapeutics.  Most of it was spent developing a cholesterol lowering drug with a Pharma company called the Medicine’s company.  In 2019 Novartis paid $9.7 billion for the cholesterol lowering drug and the Medicine’s company.)

Nature 574, S7 (2019) 

https://www.nature.com/articles/d41586-019-03070-w

https://www.pewtrusts.org/-/media/assets/2018/09/atf-transcript_episode-37.pdf

DNA is present as two connected sequences of building blocks.  Nucleotides from one strand “hold the hand” of a complementary nucleotide on the other strand.  When DNA wants to instruct a cell to make a certain protein it creates a single strand of messenger RNA.

One side of the DNA is used as a template for the messenger RNA.  For each DNA nucleotide there is an appropriate and opposite RNA nucleotide. Where there is a DNA adenosine there is an RNA thymine etc.  The side of DNA that is used as a guide is called the “anti-sense” sequence.  The RNA that is created looks like the other DNA strand –the sense side.

“In the 1990s Andrew Fire and Craig Mello were investigating the expression of a muscle gene in a nematode worm.  They injected sense, then anti sense mRNA and the behavior of the worm didn’t change. Then they injected sense and antisense RNA together, they the worms started twitching. Similar movements were seen in worms that completely lacked a functioning gene for the muscle protein.

“When sense and antisense RNA molecules meet, they bind to each other and form double-stranded RNA. They wondered if a double-stranded RNA molecule silenced the gene carrying the same code as this particular RNA? Fire and Mello injected double-stranded RNA molecules containing the genetic codes for several other worm proteins. In every experiment, injection of double-stranded RNA carrying a genetic code led to silencing of the gene containing that particular code. The protein encoded by that gene was no longer formed.”

They showed that RNA interference is specific for the gene whose code matches that of the injected RNA molecule, and that RNA interference can spread between cells and even be inherited. It was enough to inject tiny amounts of double-stranded RNA.

 (It was later determined that double-stranded RNA binds to a protein complex, Dicer, which cleaves it into fragments. Another protein complex, RISC, binds these fragments. One of the RNA strands is eliminated but the other remains bound to the RISC complex and serves as a probe to detect mRNA molecules. When an mRNA molecule can pair with the RNA fragment on RISC, it is bound to the RISC complex, cleaved and degraded. The gene served by this particular mRNA has been silenced.

RNA interference is important in the defense against viruses, particularly in lower organisms. Many viruses have a genetic code that contains double-stranded RNA. When such a virus infects a cell, it injects its RNA molecule, which immediately binds to Dicer (Fig 4A). The RISC complex is activated, viral RNA is degraded, and the cell survives the infection.”  https://www.nobelprize.org/prizes/medicine/2006/press-release/

In 1983 researchers found the first genetic disease marker.  It was linked to Huntington’s, a dominant malady that strikes in midlife.  The disorder became well known after Woody Guthrie, the Oklahoma folksinger who wrote “This Land is Your Land” learned, at age 40, that his jerky movements, rigidity, clumsiness and inability to think clearly were caused by the disease.  Abandoned at age 14 by his mother, who was hospitalized with Huntington’s, and his father, who moved to nearby town for a job, Guthrie spent his teenage years sleeping at various friend’s homes.  He was able to rejoin his dad after a few years, but was more interested in his guitar than he was in high school.  Guthrie married when he was 19 and the couple had three children.  During the dust bowl Woody was living near the Oklahoma panhandle.  Giant clouds of dust periodically blew in, filled lungs and killed cattle and a few children.  The clouds were the result of decades of farming in an ecosystem that had adapted to long droughts. Farmers had pulled out the deep seeded grass that had covered and protected the dirt for over a century. During the wet years the crops were bountiful.  Then came years when it barely rained.    The soil became hard and the winds were fierce.  Called the center of the dust bowl the area was now barely habitable and people were pulling up stakes and heading to California.  Woody decided to join them.  He left his family and headed west.  2 of Woody’s first three children developed Huntington’s in their early 40s. During his 55 years Woody served in the merchant marines, lived in California and New York and was a popular entertainer.  He married two more times and wrote 1000 songs, one of which turned out to be his final message: “so long it’s been good to know yuh.”

The nucleus of each cell in our body contains 23 strands of DNA, 23 chromosomes.  That’s where the 20,000 genes that are unique to each person are found.  These genes account, at most, for 3 percent of the DNA in each nucleus.

Over several decades researchers identified the nucleotide sequences responsible for one genetic disease, then another.  In 1990 scientists started mapping the entire human genome; the task was “declared completed” in April 1993, and genetic research got a huge boost. . We also learned that after a cell makes a protein it has to coil and fold into a specific three-dimensional shape.  Misfolding produces inactive or toxic proteins and causes a number of genetic diseases.24

Every so often a child is born with one of over 2000 really bad genetic diseases, and his or her family has to raise a disabled infant who will die young. Researchers working on the problems, have developed treatments that supply or teach the body to replace a vital protein. The number of children currently alive with each disorder is increasing.    

Companies that market these life saving products charge a lot, too much for most people.  It’s estimated that “orphan drugs will make up one fifth of worldwide prescription sales, amounting to $242bn in 2024.  Much of the money will go to either big Pharma or big biotech.2”  “The cost per patient per year of the top 100 orphan products was $150,854 in 2018.”  Insurance companies that stay in the market and ultimately the taxpayers will have a new flood of costs they will increasingly have to deal with.   

In the U.S. 49% of our health dollar is spent on 5% of the people.  The total cost of care in the 2 ½ decades between 1980 and 2004,”has gone from $1,106 per person ($255 billion overall) to $6,280 per person ($1900 billion overall).” 

There was a time when drugs for uncommon diseases had a difficult time getting FDA approval.  Tests involving large numbers of affected people were needed before the FDA would conclude a new drug was safe and effective.  That usually wasn’t possible when relatively few people were afflicted. 

Then parents got together, pressured members of Congress, and the legislature acted.  In 1983 Congress passed and the president signed the Orphan Drug Act.  Companies that manufactured drugs for less than 200,000 Americans got a lot of rewards:  Their FDA monopoly lasted seven, not five years.  Companies got tax credits—they could write off half of the development costs.  If the disease was rare, developers skipped the usual wait and joined the “fast-track” line.

 The law worked better than anyone could have predicted.  There are 7,000 rare diseases affecting 25 million to 30 million Americans. In the first 20 years 249 orphan drugs were marketed. 

The FDA has approved 3 drugs that help people with cystic fibrosis.  They were developed using seed funding from the Cystic Fibrosis Foundation –$47 million over 5 years; and $20 million from the Gates foundation. 

In kids with Cystic Fibrosis the mucous that collects bacteria and foreign particles is not watery, not easily swept out of the lungs and swallowed or coughed up.  It’s thick, “glue like”, and people with the disease have a hard time getting rid of it.  They periodically develop pneumonia, and over time they lose lung function.  A century ago most of the afflicted weren’t aggressively treated like they are now with inhaled bronchodilators, physical therapy, postural drainage, and appropriate antibiotics.  Few survived childhood.

The condition is genetic, and recessive.  If both parents are carriers, one of four offspring is afflicted. 

The Cystic Fibrosis Foundation has established 117 centers of excellence.  They are manned by experienced health care professionals and have guidelines, “best” practices, and public monitoring. As a result of their aggressive approach the average person with Cystic Fibrosis now lives an average of 35 years, though getting kids through their teen age years is typically tough. 

Vertex, a biochemical startup that spent $4 billion during its first 22 years without developing an approved drug was not anxious to get into the Cystic Fibrosis business.  There were only 30,000 Americans with the condition.  If the company found a potential drug, it would cost $100,000 to test each person. In the company’s mind the expense of getting involved was “prohibitive.” Richard Aldrich, a deal maker and advisor, thought Vertex should only work with the CF (cystic fibrosis) foundation “if the foundation agreed to fund some of the early stage (drug) development.”

The Vertex research team was headed by Eric Olson.  An experienced research biologist, he was interested in CF.  A Colleague/friend’s daughter had the disease.

The faulty piece of DNA, the cause of the disease, was located in 1989 by a group of Canadians geneticists working with Hong Kong born Lap-Chee Tsui.  The mutation responsible for most cases of cystic fibrosis occurs when three nucleotides are deleted from a gene on chromosome 7.  Called Cystic fibrosis trans-membrane conductance regulator (CFTR), the abnormal gene causes the cell to make a defective membrane protein, one that doesn’t “fold” normally.  Appropriately folded protein regulates the amount of chloride, salt and water that flows in and out, of the cell. The fluid travels through “channels” in the cell’s outer wall or membrane.  In people with Cystic Fibrosis, salt accumulates outside the cell and secretions are thick. Sweat is salty. 

By June of 2011 Vertex had two drugs that dropped the salt content of sweat.  They decreased the exacerbation rate, and the unexplained “worsenings” that contributed to a more rapid decline in pulmonary function.  In the early 2000s Vertex added a third drug.  It had an effect people who have a “Phe508del  CFTR  mutation.” It’s the most common abnormal gene.  90 percent of people with cystic fibrosis have at least one copy of the mutated DNA.  One analyst felt the triple-drug combo will rake in close to $4.3 billion by 2024.

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The most common genetic cause of death in infancy, Spinal Muscular Atrophy “causes severe weakness by 6 months of age and inability to breathe by the age of two.”  The gene that directs cells to make an essential protein is deleted or mutated.  In the absence of the protein, the nerves that send signals to muscles die. 

We all have a second gene that’s similar and that isn’t genetically affected.  But it doesn’t work.  It’s not able to make the needed protein.  And that’s OK because most of us don’t need it.

Scientists at Cold Harbor Laboratory, a huge nonprofit, developed a “segment of RNA” that, when injected into the spinal fluid, allowed the second gene to make the needed protein. It was a great accomplishment and it wasn’t easy.  The chemical, called nusinersen, was developed with the assistance of researchers at Isis pharmaceuticals. Another for-profit firm, Biogen Pharma, paid for the testing.  Once nusinersen was shown to be effective Biogen paid millions and bought everyone else out. When the drug was approved by the FDA its owner decided to charge $125,000 for each dose, or $750,000 the first year and half as much each subsequent year.27 

  A physician at the University of Utah who cares for “about 150 patients with the disease, complained in an article that if each child was treated with nusinersen, the cost would be $113 million the first year and $56 million thereafter.10”

In November 2017 an Ohio company developed alternative approach to the problem.  Their therapy was based on research performed at Nationwide, a Columbus Ohio children’s hospital, by Brian Kasper.  An employed researcher, he studied adeno-associated viruses (AAVs).  (He presumably was learning how to put plasmids that contained genes into harmless viruses.  The viruses would then infect a body, enter its cells, and dump the plasmids.)

One day his team discovered a viral serotype that penetrated the blood brain barrier. There are 50 serotypes of adenoviruses.  They don’t usually make people sick, and most can’t get into the brain.

Kaspar and team believed they had “a new way of delivering genes to widespread regions of the central nervous system.  The drug companies they approached allegedly weren’t interested.  So in 2013 with the help of a biotech entrepreneur, Kasper formed a startup, AveXis.  They raised $75 million and licensed the therapy from the Columbus hospital.  To this point all research and development was paid for by the U.S. government and charitable funds.

Researchers placed a gene that promoted the production of the needed protein into an adenovirus.  I presume that the SMN1 and SMN2 genes in plasmid form were available and could be purchased from Addgene for $65. 

They infused a high dose of the virus that contained the gene into the bloodstream of 12 affected children who were about 6 months old.  After 1 ½ to two years 11 of the children were able to speak, 9 could sit unassisted for at least 30 seconds, 11 achieved head control, 9 could roll over, and 2 were able to crawl, pull to stand, stand independently, and walk independently. The research was presumably paid for by some of the startup’s $75 million. 12

In April 2018 Novartis bought Avexis for $8.7 billion.  After the FDA approved the therapeutic approach, Novartis named the treatment Zolgensma.  In an attempt to recover their multi-billion dollar investment, and make a profit the Swiss set a high price for a treatment.  Novartis will charge each child or their insurer $2.125 Million.29

Currently “there are more than 800 cell- and gene-therapy programs in clinical development. Several of these therapies have been approved by the FDA.”—And the science is in its infancy. Some of the treatments on the market are owned by Biomarin, a company headquartered in San Rafael California.  Founded in 1997 the company has acquired 6 biomedical startups in the last 15 years.  In 2016 were marketing 5 orphan drugs.

When a tear occurs in a blood vessel people bleed, platelets plug the hole, and a sequence of proteins pile on.   A clot won’t form if the person’s serum doesn’t contain enough clotting factor 8 or clotting factor 9.  People who genetically don’t make sufficient amounts of either of these proteins have hemophilia.

A genetic condition that “occurs in approximately one in 5000 live births,” hemophilia is sex linked– which means that women carry the gene and their sons get the disease.  When injured, affected males whose factor 8 or 9 is low don’t stop bleeding.  Their joints periodically and very painfully fill up with blood.  Over time they develop joint deformities. 

Victoria, the queen of Great Britain from 1837 to 2001 was a carrier of the hemophilia B gene.  She passed the condition through her daughter Alexandra to her grandson Alexei, the only son of Russian Tsar Nicholas. The couple had 4 daughters.  The boy’s painful and frightening bleeds seemed to be helped by a self proclaimed holy man named Rasputin.  During the First World War Tsar Nicholas spent a lot of time at the front, and his wife was in charge of the government.  Much to the chagrin of the Moscow elite she seemed to be “under the spell” of the holy man. 

The war went badly for Russia.  Over 5 million soldiers were killed or wounded.  After two years the Russian people had enough and they rebelled.  They deposed the Tsar and Russia withdrew from the conflict.  Some believe that hemophilia and the power of the mystic played a big role in 1917 fall of the empire.26

Men whose blood level of clotting factor 8 is at or below one percent have a severe condition.  Those whose blood levels are 5 to 40 percent have a moderate problem and mainly receive factor 8 infusions before surgery or if there is a need.14 

In the 1980s, during the height of the AIDS epidemic, small amounts of the factor that stopped hemophiliacs from bleeding was collected from each of hundreds of units of plasma that was obtained from donors.   One of the units usually came from a person who had HIV but didn’t know it.  When the young men with hemophilia received the contaminated factor they developed AIDS, a disease that, at the time, was lethal.

Researchers at the University College London recently put a portion of the factor 8 gene into an adeno-associated virus and “infected” a number of men.  With the gene floating in their cytoplasm, cells in the liver made the missing protein. In 6 of 7 patients receiving high doses of genes “factor 8 increased to a normal level and stayed there for a year.  None of the 7 bled during that time.”   After 2 to 3 years the treatment was still providing a clinically relevant benefit.21”

10 men with hemophilia B whose blood had less than 2% of the needed clotting factor were infused with an adeno virus containing a replacement gene.  During the subsequent year their clotting levels rose and stayed at a mean level of 33 %, bleeding virtually stopped, and only 2 patients needed a factor infusion. 

Scientists seem to be getting close to solving hemophilia.

As explained on 60 minutes, “Francis Collins of the NIH thinks we can cure sickle cell anemia by using CRISPR gene editing to increase blood levels of fetal hemoglobin (HbF).   HbF is the form of hemoglobin that fetuses use to efficiently extract oxygen from the placenta and deliver it to their bodies. Shortly after birth a gene causes most children stop producing Hemoblobin F and adult hemoglobin takes over.    

People with Sickle Cell disease have a genetic abnormality that affects adult hemoglobin. Red cells that should be round and flexible start looking like sickles or crescent moons.  They clump, stick in small blood vessels, and cause severe pain, anemia, stroke, pulmonary hypertension, organ failure, and far too often, early death.

Researchers at Vertex and CRISPR Therapeutics collected stem cells from a person with severe Sickle cell disease.  In the lab they used CRISPR to destroy the gene in the stem cells that shuts down production of fetal hemoglobin. Then they destroyed the remaining bone marrow with chemotherapy and infused the edited cells into the patient.23 It seemed to be working.18

CRISPR derived gene therapy is new, exciting and not fully developed, but will be widely used in future gene editing. The investigators who did many of the studies and developed the concept were publically funded and were—at the time—trying to learn how bacteria defend themselves from assaulting viruses.  They were trying to understand CRISPR.

The following theory of how CRISPR came into being helped me understand the process: When a virus assaults a bacteria, the invader enters the cell, takes over its DNA, and directs the bacteria to make billions of viral particles.  Most bacteria are enslaved, then destroyed.  A few mount a defense and survive.  Some of the survivors create a “DNA memory file.”  The identifying characteristics of the bad viruses are stored in the DNA’s CRISPR area, and the memory–sequence becomes a “gene” that is passed on to future bacteria.  In subsequent generations the memory DNA creates strands of RNA that float around inside the bacteria.  When a segment of RNA recognizes an invading virus it latches on.  Then it cuts the virus apart with an enzyme called Cas9.

After they understood how bacteria identify and destroy unwanted viruses, researchers tried to use the system to edit genes.  They chose a target–a “twenty-letter DNA sequence” that was part of the gene they wanted to delete. Then they “converted” a collection of nucleotides “into a matching 20 letter strand of RNA.”  This RNA was an exact replica of the DNA.  It would guide the Cas-9 past the cell’s 3 billion pairs of DNA nucleotides and it would eventually identify the desired strand of DNA.

They planted the genetic instructions for making Cas9—the knife—into one plasmid. They put the genetic instructions for “guide RNA” –-into a second plasmid.

Their concoction was able to search a cell’s DNA—3 Billion pairs of nucleotides—and find the desired segment of DNA–and unwind the DNA –and use Cas 9 to cut apart the strand of nucleotides.  In other words they could irreparably damage a chosen gene.  (Cells know how to repair a break in their DNA. The cut ends either come together on their own.– Or the gap can be bridged by a segment of DNA.)

The study group was led by UC Professor Jennifer Doudna and Emmanuelle Charpentier.  Doudna, the daughter of a professor of English, grew up in Hawaii and spent the summer that followed her college freshman year in a lab studying a fungus that was invading papayas.  “It turned out to be a lot of fun”, she hungered for more, worked in a few labs, made a few discoveries.  After a decade she became the head of a research lab at the University of California in Berkeley, a campus that was often blanketed in fog, but on clear days provided a spectacular view of the Golden Gate Bridge and the Pacific.

Doudna met Emmanuel Charpenier, a French professor who was working in Sweden, at a 2011 conference in Puerto Rico.  Described as “Small and slight, with eyes so dark that they seem black” Charpentier was a PhD student at the Pasteur Institute in Paris when she “realized she had found her environment.”   After that she spent more than 20 years performing research in 9 different institutes in 5 different countries.29

The two women discussed possibly collaborating while they explored the narrow cobbled lanes of old San Juan.   

In June 2012 Doudna and Charpentier published a study that showed how RNA and Cas9 could be used for “site-specific DNA cleavage and RNA-programmable genome editing”.  Investigators around the world took notice and got busy.

Scientists already knew how to add a new, good gene.  Mario Capecchi, years back learned that genes intuitively know where, amidst the 3 billion pairs of DNA nucleotides, they belong.  If good genes are developed and put into cells, they migrate and attach to “their place.22.” 

After Doudna’s paper was published Kevin Esvelt (currently at MIT) explained how using CRISPR + selfish genes in the germ line can create changes that will be inherited by future generations of cells.

Sensing that there wasn’t time to write grants and get government funding, Doudna and other scientists formed a venture capital company– Editas Medicine.  Charpentier and others founded CRISPR therapeutics.  Both firms, according to their web sites, are trying to cure Sickle Cell disease, cystic fibrosis, and a few other genetic conditions.19

Medical thinkers have argued that:  given our current experience with the price of drugs we should start thinking about how we’re going to pay for “future success in gene therapy.”20

  1. Wurm FM (2004). “Production of recombinant protein therapeutics in cultivated mammalian cells”. Nature Biotechnology. 22 (11): 1393–1398. doi:10.1038/nbt1026. PMID 15529164. 
  2. Orphan Drug Report 2019 https://info.evaluate.com/rs/607-YGS-364/images/EvaluatePharma%20Orphan%20Drug%20Report%202019.pdf?mkt_tok=eyJpIjoiWWpVMk1UVmtNRFpqT0dFeiIsInQiOiIrcmZ3QjNwamZWWVwvZ1ZkcU5XS2E3Rk5oNXA5MXZJVUVCRitMQXpQd0sxMGJPU0JhdGRWbVJQQkZrc0xZNDNPSXRNM09wMGh2OEFXNXFNN1wvb1plT
  3. https://archive.ahrq.gov/research/findings/factsheets/costs/expriach/  http://www.paradigmglobalevents.com/events/orphan-drugs-rare-diseases-2017-americas/
  4. (Victoria Sato, president of Vertex– Page 69). Barry Werth, The Antidote. Simon and Schuster, 2014
  5. http://www.nejm.org/doi/full/10.1056/NEJMe1712335
  6. (Science: Dec 20, 2019)     https://www.nejm.org/doi/full/10.1056/NEJMoa1908639
  7. John Cohen, Science. Page 130, January 10, 2020
  8. https://www.nejm.org/doi/full/10.1056/NEJMoa1908639?query=featured_home  https://www.fool.com/investing/2019/06/12/5-most-valuable-pipeline-drugs-in-development-and.asp
  9. http://btn.com/2017/06/10/how-iowa-and-rutgers-are-taking-down-a-rare-and-devastating-disorder-btn-livebig/
  10. N Engl J Med 2017; 377:1723-1732 https://hbr.org/2017/04/the-cost-of-drugs-for-rare-diseases-is-threatening-the-u-s-health-care-system
  11. NEngl J Med 2017; 377:1713-1722. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900005/
  12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895694/   Nat Biotechnol. 2009 Jan; 27(1): 59–65. Crystal, Ronald g. Adenovirus: The First Effective In Vivo Gene Delivery Vector Hum Gene Ther. 2014 Jan 1; 25(1): 3–11.  https://www.nejm.org/doi/full/10.1056/NEJMoa1706198
  13. https://xconomy.com/national/2019/04/15/how-an-ohio-kids-hospital-quietly-became-ground-zero-for-gene-therapy/
  14. High, Katherine, N Engl J Med 2019; 381:455-464 . N Engl J Med 2020; 382:29-40  https://www.nejm.org/doi/full/10.1056/NEJMoa1708483
  15. https://www.nejm.org/doi/full/10.1056/NEJMra1706910
  16. NEJM Aug 1, 2019.    NEJM Dec 7, 2017.  https://www.nejm.org/doi/full/10.1056/NEJMoa1708538
  17. N Engl J Med  May 14, 2015   https://www.nejm.org/doi/full/10.1056/NEJMoa1414221 6 http://www.cnn.com/2017/10/12/health/fda-gene-therapy-blindness-vote/index.    html https://www.forbes.com/sites/matthewherper/2017/12/11/spark-shadows-biomarin-in-hemophilia-gene-therapy-race/#7af39d156106  http://www.ucl.ac.uk/news/news-articles/1217/131217-UCL-research-leads-to-haemophilia-therapy-success
  18. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870126/   Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat Med. 2019 Mar 25.  CRISPR Therapeutics and Vertex Announce FDA Fast Track Designation for CTX001 for the Treatment of Sickle Cell Disease , CRISPR Therapeutics News Release, Jan. 4, 2019.   https://www.nhlbi.nih.gov/news/2019/nih-researchers-create-new-viral-vector-improved-gene-therapy-sickle-cell-disease-0  )
  19. A Crack in Creation by Jennifer Doudna and Samuel Sternberg:  Mariner Books.  2018.
  20. (“Stuart Orkin, MD, and co-author Philip Reilly, MD, JD, of Third Rock Ventures, tried to “catalyze the discussion” by suggesting several new models for valuing, pricing and developing gene therapy.”) Orkin, P. Reilly. Paying for future success in gene therapy. Science, 2016; https://www.nejm.org/doi/full/10.1056/NEJMra1706910
  21. (After three years:   2 –who received a lower gene dose (2 x 1013 or less) had very low levels of factor 8–1 IU per deciliter.   7—who received a higher dose (6 x 10 13  ) had a median factor VIII level of 20 IU per deciliter;  None of the 7 bled and none were treated with factor 8.  (Before treatment the 7 were receiving 138.5 infusions annually.)  (6—who received a median dose (4 x 10 13  ) were followed for two years.  At the end of that time the median factor VIII level was 13 IU.  None of the 6 bled, but three had received one infusion of factor 8 per year.  (Before treatment the average person was getting an infusion every other week.15)
  22. (Mario Capecchi of the University of Utah made the discovery in 1982, and won the Nobel prize in 2007).
  23. Company claims signs of success with CRISPR-edited stem cell transplants for two genetic diseases By Jon Cohen Nov. 19, 2019, Science.
  24. http://news.mit.edu/1993/huntington-0331
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  3. Nicholas and Alexandra by Raymond Massie. Random House ; 1967
  4. .  https://smanewstoday.com/2015/02/23/exclusive-new-insight-into-sma-from-dr-adrian-krainer/ 
  5. Gene therapy for spinal muscular atrophy  https://www.nejm.org/doi/full/10.1056/NEJMoa1706198
  6. Charpentier https://www.nature.com/news/the-quiet-revolutionary-how-the-co-discovery-of-crispr-explosively-changed-emmanuelle-charpentier-s-life-1.19814
  7. Amyloidosis https://www.sfgate.com/health/article/The-Domino-Effect-Woman-gets-new-liver-gives-3238583.php