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Ronald G. Crystal, M.D.
Now let us turn to another of the early applications of gene therapy in the clinic, to the genetic disorder cystic fibrosis. Dr. Ron Cyrstal is Chief of Pulmonary and Critical Care, Cornell Medical Center and Professor of Physiology and Biophysics, Cornell Graduate School of Medical Sciences.
Dr. Crystal, how did you first become interested in gene therapy?
Ron Crystal: Our laboratory had been interested for a number of years in a hereditary disorder associated with emphysema called Alpha I Anti-trypsin deficiency. Alpha I Anti-trypsin, a protein produced by the liver, is the second most common or abundant protein in the blood and when you have a deficiency of Alpha I anti-trypsin which occurs when you have mutations in the two peripheral genes, there is a deficiency in Alpha I Anti-trypsin in the blood and hence throughout the body. Alpha I Anti-trypsin functions as an anti-protease that protects the lung from proteolytic or enzymatic destruction from white blood cells. If you don't have enough Alpha I Anti-trypsin, white blood cells over years injure the lung and cause emphysema. We had developed a therapy for Alpha I Anti-trypsin deficiency by purifying the protein from blood and then went on to carry on studies that went to the Food and Drug Administration approval of Alpha I anti-trypsin replacement therapy.
You had a successful conventional therapy for Alpha 1 Anti-trypsin why did you turn to gene therapy?
Ron Crystal: In the mid-80s,we started getting interested in the idea of rather than giving back the protein that was deficient why not have the body produce it by taking the gene and putting it into the individual that was deficient. The first strategy was to use a retrovirus vector and put fibroblasts, showed that the fibroblasts could produce Alpha I A trypsin. We then took those modified fibroblasts and put them in the peritoneum of experimental animals and demonstrated that we could detect the Alpha I a-trypsin in the blood in the womb. So we knew that worked. The problem is we couldn't get enough. It's a very abundant protein and so we knew that that strategy using retroviruses in the so-called ex vivo approach to gene therapy just wouldn't be a sufficient treatment of the disease. We went on to use retroviruses and put them in lymphocytes and put the lymphocytes down the lung but we still couldn't get enough.
What steps did you take to increase levels of the enzyme?
Ron Crystal: In the spring of 1989,, I became aware of the possible use of group C serotype II and V adenoviruses as possible gene transfer vectors. In April of 1989 we started working with the adenovirus that was deleted in the E-I region, that is the region that controls the ability of the adenovirus to replicate. By taking that out and putting in its place the Alpha I Anti-trypsin CDNA with appropriate promoter we began looking at the ability of using this for in vivo gene therapy, that is where we directly administered an adenovirus expressing of I a-trypsin to the experimental animal. This led to a paper in 1991 in Science that described that strategy where we directly infected lung epithelial cells in vivo with an adenovirus coating for Alpha I anti-trypsin. The concept was that the epithelial cells lining the lung would produce the Alpha I anti-trypsin and then secrete it into the lung. In fact it does and it works. Again the problem was you don't quite get enough and so we had to make a decision as to whether we pursue that in terms of moving it towards humans or do we go in a different direction.
It was at this point that you turned to cystic fibrosis?
Ron Crystal: We had been very interested in the possibility of using adenovirus for gene therapy for cystic fibrosis. In fact that's how we got the first idea to use adenoviruses because we knew as pulmonary physicians that adenoviruses were trophic for the epithelium of the human airwaves ---- infects the cells of the upper and lower respiratory tract and so we put the cystic fibrosis normal gene, referred to as the cystic fibrosis transmembrane conductance regulator gene, into an adenovirus vector. We were able to demonstrate that we could effect the airway epithelium of experimental animals and that led to a paper in Cell in early 92, but by that time we knew that it certainly was logical to consider using this kind of strategy to try to transfer genes to humans with cystic fibrosis.
What regulatory hurdles did you have to overcome before you could start these clinical trials in human beings?
Ron Crystal: We proposed that to the appropriate regulatory agencies including the Food and Drug Administration and the DNA Recombinant Advisory Committee as well as appropriate institutional review board and moved ahead so that by April of 1993 we carried out the first human gene therapy studies using a virus directly in the lung and these were for individuals with cystic fibrosis.
You must be able to demonstrate that the gene is being expressed how do you do that?
Ron Crystal: In terms of testing these vectors, the strategy that we use for assessment of in vivo gene therapy is primarily to assess expression in the target cells, in this case the airway epithelium. The way we do it now is we use a fiberoptic bronchoscope and we spray the adenovirus vector expressing the cystic fibrosis transmembrane conductance regulator cDNA, directly onto the airway of the epithelium. Then we go back with a bronchoscope and a fine little brush and we brush the epithelium and we recover millions of cells and then we use quantitative polymerase chain reaction technologies to quantify the amount of CFTR, cystic fibrosis transmembrane conductance regulator, message RNA driven off the vector compared to endogenous CFTR messenger RNA. The results to date have demonstrated that we can get levels of vector driven message RNA safely that are at or above the levels that we believe are necessary to cure the disease. The problem is that it goes away. We don't know how long it lasts exactly. We know we can get normal levels three days after administration of the vector in humans with cystic fibrosis, but by 30 days which is the next time we measure it, it's gone. If we try to readminister we can get it again but it's a little lower, but it goes away again. By the third time we try administer we cannot get expression.
What do you believe is going on?
Ron Crystal: From animal experiments we believe that those are host defenses against the adenovirus that are preventing from repetitively administering it with successful expression on a persistent basis. So we are in the midst of evaluating a variety of second, third generation adenovirus vectors, alone and together with immunosuppressive agents. The idea is to try to either trick the immune system or suppress the immune system or more likely both to try to get expression to persist. Cystic fibrosis is a hereditary disease so we need expression on a persistent basis.
How are you trying to overcome this problem of the patients immune response destroying the activity?
Ron Crystal: We became so frustrated about a year and a half ago about the lack of correlation between experimental animal studies, particularly in mice but other animals as well, not giving similar results as we saw in humans with cystic fibrosis that we decided to work host defenses against these viruses. Whether or not our new generation of vectors would really work, that would be much more straightforward to work it out in normal individuals rather than in individuals with cystic fibrosis. Our strategy is to evaluate the first generation vectors, the newer generation vectors that we developed as well the immunosuppressive agents in a variety of strategies where we're looking at host responses as well as persistence of the vector. As we begin to see advantages for some of these new generation vectors and or with immunosuppressive agents or strategies then to move that into individuals with cystic fibrosis to see whether or not we can attain the kind of persistence that we need.
We have been discussing using adenoviruses to deliver genes to the lungs. Can they be used for other tissues?
Ron Crystal: About three years ago, together with my colleague Ted Rosengard in the Department of Cardiothoracic Surgery here at New York Hospital, Cornell Medical Center, we began thinking about how we could use adenovirus vectors that are available today for a problem that is significant and that is the problem of cardiac eschemia. We've developed an adenovirus to express the vascular endothelial growth factor cDNA. We carried out a series of studies in experimental animals to show that in fact we could use this to make new blood vessels. Vascular endothelial growth factors is one of the ways the heart normally uses to make blood vessels when it's growing in an embryo and growing through the neo-natal period and in childhood. When we get to be adults our hearts sortof forget genetically how to make new blood vessels. So the idea was to directly administer an adenovirus to the heart with the idea of getting the cardiac ________sites to produce vascular endothelial growth factor to tell the blood vessels in the local milieu to grow and to bypass areas of obstruction. Experimental animal studies dramatically demonstrated that in fact it worked and after doing a series of safety studies in December of 1997, Dr. Rosengard and I initiated human trials where we directly administer the vector to the heart in the area of the schemia. These studies are progressing and if they track the way the experimental animal studies track then we are very hopeful that this may evolve into a successful therapy. We of course will have to wait for the assessment of the studies to determine how successful it will be.
Would you like to close with your general view of the field of gene therapy?
Ron Crystal: We do some of our gene therapy studies on cystic fibrosis and normals at the Rockefeller University where my colleagues and I also work which is right next door to the New York Hospital Cornell Medical Center. We do our gene therapy studies one hundred feet from where Avery and his colleagues discovered that DNA was the genetic material in the early 1940s. This is remarkable when you think that in the early 1940s science did not even know that DNA was genetic material and now, through the human genome project we will have all 70 to 100,000 genes that make us up as humans on our PCs, we'll have all the sequences. Now we have the technology through gene therapy not only with adenovirus but with other vector systems that we clearly can deliver genes to humans and do so safely. We may not be able to do it on a persistent basis or in the amounts we want or exactly how we want to do it yet but clearly the field has moved ahead very, very rapidly. The other remarkable thing thinking back is that we did the first human studies using a virus to transfer a gene in vivo in 1993 and here we are just five years later and we are doing gene transfer studies in normal individuals. We are directly administering these vectors directly to the hearts of humans to make new blood vessels. I think that that, together with all the other really exciting developments by many investigators throughout the world in the gene therapy field, suggests that we are embarking on what at least I and my colleagues in the field believe will be a revolution in therapeutics. It may take several years to develop. It may not be with the vectors that we think are going to be successful now. It may not be exactly the way we are all working, but together with the information of the human genome and studies in functional genomics, gene therapy clearly is going to be a major paradigm for therapeutics for the future.