Cancer Gene Therapy

>>> good afternoon. i'm the deputy director of the nhlbi and i'm delighted to welcome dr. kathy high as our wals lecturer today. she is the director of the center for cellular and molecular therapeutics at the children's hospital in

philadelphia. an attending hemetologist and the william h bennett professor of pediatrics at the university pennsylvania school of medicine and a howard hughes institute fellow. last month, kathi and fredrico published a review article in

the journal, blood, entitled, "immune responses to aav vector, overcoming barriers to successful gene therapy." this is an elegant survey of the state-of-the-art gene survey, using aav vectors. gene therapy products for the treatment of genetic diseases

are now currently in clinical trials and an aav product has been licensed in europe. the aav vectors have achieved positive results in a number of clinical and preclinical settings, including homologic diseases, such as heme philias and hemochrometosis and others.

this is an extremely hot topic and we are delighted to have catherine here. aav vectors are administered to the patient and therefore listed as a host immune response and this is actually a major topic of dr. high's current research. she is trying to achieve a

comprehensive understanding of the determinants immunogenicity of these aav vectors and the potential associated toxicities. in collaboration with global leaders in the field, she is engaged in immunosurveillance of ongoing clinical studies to provide the basis for

understanding of the i want keys of the immune response in aav mediated gene transfer facilitating safe and effective therapies for genetic diseases. kathy has an interesting career trajectory. she majored in chemistry as an undergraduate and spent a year

working in the biology of arthrosclerosis in college. this led her to pursue an md instead of a ph.d. in chemistry as she had originally planned. but we almost lost her to medicine because the emphasis on memorizing facts as a first year medical student discouraged her.

she took a year off to do chemistry research during which her lanmentor in chemistry encouraged her to finish medical school. fortunately for us, when she returned to medical school, things clicked and she decided to stay in medicine, but her

solid grounding in science and chemistry served her well. kathy's work regularly joins the boundaries between the bench and the bedside. she was drawn to hematology because of the extremely sophisticated understanding of the biology of many homologic

diseases on a molecular level. this matches her interest in biochemistry, chemistry and molecules. as she was completing her hematology fellowship at yale, scientists cloned the genes for factor 8 and factor 9. this ft. presented her with an

opportunity to sort out the molecular basis of disease utilizing her chemistry training and her tremendous interest in the biochemical origins of disease. her initial work focused on defining mutations and clotting actor genes that result the in

hemophilia leading to ability to identify carriers and permit prenatal councilling to women who carry the mutation and allowed her to analyze the relationship of the structure of the molecules and their fung. kathy and her colleagues developed models of hemophilia

in mice and dogs that work with dogs in which heme philia occurs naturally. they demonstrated they can successfully treat the disease in mice and dogs by inserting a normal clotting factor gene into the animals. she is now focused on adapting

therapies for humans. her attempts at gene therapy have focused primarily on heme philia b, the less common form of the disease, effects 3000 men and boys in the united states. she demonstrateds in dogs with heme philia b the principle that gene therapy can be effective in

treating the disorder. dogs that receive a single injection for the gene for factor 8 produce the clotting factor for more than five years. human trials proven to be a little bit more challenging. in small clinical trials using an aav vector, a few patients

produce therapeutic levels of clotting protein but only for a few weeks. she is now exploring ways to modulate the patients immune response long enough for the virus to deliver the gene safely to the cells. her major areas of investigation

include the structure funds analysis and factors 7, 9 and 10, recombinant mutations and the regulation of expression of genes. please welcome dr. kathy high who will talk to us about genetic therapies for genetic disease, results and lessons

from recent successes. i'd also like to remind you we have a reception which is sponsored by the faes in the library directly after the lecture and to invite all of you to attend. kathy? [ applause ]

>> i certainly like to thank dr. sher in for that wonderful introduction. i have to say that my career sounded much more interesting as she told it than it seems to have been during the living of it. at least to a certain extent.

i thought that what i would do today, because this is an nih-wide lecture, is talk to you not ole' only about some of the hurdlingss that we encountered in attempting to work out the details of gene therapy for heme philia, but also talk a little bit about the work that we have

done in collaboration with the alpha managey group at the university of pennsylvania of conducting a trial for a rare inherited form of blindness. and then if there is a little time at the end, i'd like to say a little bit about the work we have been doing most recently

using genome editing to approach treatment of genetic disease. so i'll just remind you at the outset that i work at a children's hospital and that genetic diseases accounted for anywhere from 30-50% of all admissions to children's hospitals and even for a very

substantial proportion of admission to adult hospitals and for many of these disorders our therapeutic ones are fairly limited and one of the hopes of the human genome project was that it would provide a platform from which we could develop gene therapies to expand therapeutic

options for people born with genetic disease. the goal of all gene therapy studies is essentially two-fold in the setting of genetic one is to achieve long-term expression of the donated gene and the other is to express it at levels high enough to have a

therapeutic effect. and all gene therapy strategies can be described essential neterms of three components, first the gene to be transferred in, the transgene, which in the case of genetic disease is not usually under dispute in contrast to the situation in

many acquired disorders. of course, in addition to supplying the missing gene, one can do variations of this strategy where you supply instead a downstream gene product or something that enables an alternate pathway, for example, 7a for somebody who

heme philia. but in general, this is spelled out clearly in genetic disease and then a target tissue into which it is introduced. so for example, hematopoietic cells for sickle cell anemia. to achieve long term expression, one can choose from two

different strategies. son to use integrating vector and introduce it into a stem cell and from there, it is pasted on to every daughter cell and one can achieve long-term expression on that basis. the other strategy, which is the one that we have been pursuing,

is to transduce long-lived post mitotic cell types. these are cell types in which, as long as the donate the dna is stabilized, it does not necessarily need to be integrated. one can achieve long term expression simply because the

cell itself is long lived and is not going to divide. so, as you may guess from the picture here, many of these strategies using integrating vectors used as their target, hematopoietic stem cells lend themselves well to being extract friday a patient manipulated in

the laboratory and returned to the patient. whereas most of these long-lived post mitotic cell types, including cardiac muscle skeltal muscle, cells in the brain and the eye do not lend themselves well to being extracted from the patient and returned but rather

require the vector be delivered in vivo. so, that of course, spells or predicts one of the major problems, which is that if you are going to introduce a viral vector directly into the patient, you're going to be grappling at close quarters with

the human immune response. so, all of our work has been with an aav vector and these were or have been engineered from a member of the parvo virus family. these are nonenveloped viruses with linear ssdna genome of 4.7 kilobases.

there are a number of features that make it attractive as a gene delivery vehicle. it would be fair to say of all viral vectors, it's one of the simplest, which is generally a positive feature. as a vector engineered for gene delivery, it is nonpathogenic

because wildtype aav is nonpathogenic. it is naturally replication defective, again because wildtype aav is wildtype replication defective. and the dna introduced aav vectors is stabilized redomestically in a

nonintegrating form. so problems are minimized -- predominantly. in the mid 90s two different groups described the result, recombinant aav vect error introduced into skeletal in mice and has become clear that aav vectors in their naturally

occurring serotypes and in genetically engineered forms as well, have topism for a wide variety of cell types, including a number of post mitotic cell types that are long lived. so the wildtype virus in codes -ine codes two types of genes required for replication

and those that encode the caps id. the veer yon is composed of essentially a wildtype caps id which has 48 molecules of viral protein 3 and 6 each of vp1 and 2 all encoded in this caps are id sequence. eeee caps id sequence.

the viral genome is replaced with the therapeutic gene of interest under the control of a appropriate promotor. so it is composed of 74% of protein and 26% of dna. and so what you centrally have in this recombinant veryon is a highly-ordered set of proteins,

the capsid that enclose the dna, the therapeutic agent. this material is dosed in vector genomes per kilogram of body weight of the recipient and since it is pretty difficult to fight physics, you can't package more than about 5 kill bases of dna into the aav capsid.

and there are a lot of different ways of producing aav. this highlights the method that we use at children's hospital and essentially, three different plasmids, one encoding the gene of interest cloned between the -- and the third providing the adenoviral helper genes

needed are introduced into 293 cells and subsequently the recombinant veryons can be harvested from the supernatin or from the cell lice aid, and one can either use all of the product purified from the cell lice aid or separated into the fulls and empties, which still

constitute a substantial proportion of what is generated in most production methods. i want doob sure to say that the first gene therapy product in ich country simulator, if you don't know what that means, it means the united states, europe

or japan, the first aav product was licensed last year in europe. this is a product for likeo protein lipase deficiency and i think it is important to point out that this product is based very heavily on work done here at the nih by rob cotton and his

colleagues. as dr. shurin mentioned, i have a longstanding interest in hemophilia. and i have been working for a number of years to try to develop an aav gene therapy for hemophilia and since colonic factors are normally made in the

liver, liver has been an important target for us. a number of obstacles had to be overcome to make aav gene in the liver a reality. we could make enough vector to do mice in the orlandoy days but not to do hemephilic dogs. in the initial trials when it

was introduced into liver, we encountered the problem, that had not been foretold by animal models and that was that the vector was detectedded in the seamen of the first subjects and had to work out strategies to mitigate the risk to track that and reduce risk of a piece of

vector dna being incorporated into an offspring. i'm going to talk about the issue that is resulted from the human immune response and for any new class of therapeutics, one issue that must be addressed is what is the long-term safety related to the use of the

product. and for aav, issues are likely at or about related to integration. but any time you introduce dna into a cell, there is a possibility that some of it will integrate into the host cell genome.

this is true for aav as well and the question is, will this have long term effects? -- had to be solved to make gene therapy for hemophilia they are a highly ordered set of proteins that express receptors on the cell surface and deliver dna to the nucleus.

the delivery proteins are degraded and get long term expression and that was observed over and over again in many large animal models before human trials were conducted. however, aav vectors viewed from an immunologic standpoint have been engineered to understand

exactly what are the human immune dose responses and get a therapeutic outcome. so i would say that our lab, over the last several years, has been focused on the following questions from a biological standpoint. what host immune responses

encountered by recombinant veryon that fails to synthesize viral antigens and fails to synthesis the viral genes that normally down regulate a host immune response and from a medical and therapeutic standpoint, can the host immune response that is generated by

recombinant veryon be controlled so the gene transfer occurs in the host is unharmed. and this is made more complex for the fact that every target issue, the human immune response is tissue-specific. so the answer to these questions is essentially tissue-specific

answers. you have to work it out one tissue at a time. so, let me just say a word or two about hemophilia. it's the excellent leading -- bleeding disorder that arises because of mutations either in the gene for factor 9, an

enzyme, or for factor 8, the co-enzyme for the same step in coagulation. and that clinically, these are indistinguishable and are characterized by frequent bleeds into the joints soft tissues but patients, especially those who are severely effected can have

bleeds into other closed spaces such as intracranial space where the bleeds can be rapidly fatal. hemophilia a effects about 1-5000 male births and hemophilia b1-30,000 male births. the other part that is important is this, the largest category of

patients with both diseases are severely effected with less than 1% factor 8 or factor 9. but we know from the natural history of the disease that if the circulating levels are even slightly better, the patient is spared most of the life threat knowing bleeds and many of the

joint bleeds as well. and if a person's level is raised to around 5%, this person may go through childhood without ever being diagnosed. so a relatively mild phenotype. we know for what we are attempting to do, that if we can elevate the levels by just a few

percent, we are likely to improve the phenotype of the and right now, of course, hemophilia is treated by intervenous infusion of clotting factor concentrates with plasma derived or recombinant and typically children are treated in a clinic until they reach the

age of about 4. you get these infusions two or three times a week and then starting at around 4, the parents can be taught to do the intravenus infusion and subsequently children learn to do it themselves. these products cost around

200,000 dollars a year for an adult male who is infusing himself 2-3 times a week. and because of these costs, it's estimated they are available for about 20% of the world's hemophilia population. if you look at the rest of the world's hemophilia population

based on a per capita gnp, this graph guess plays children with hemophilia compared to adults with hemophilia as a function of the gnp. and in the western world, there are many more adults than children. but then as the gnp falls, it

really becomes progressively a lethal disorder that kills most people before they reach adulthood in resource-poor nations. so, i'll skip over a lot of preclinical work that we have other people have done in mice and dogs and say that by the

late 1990s, we have the following result in the naturally-occurring hemophilia b dog model that a single injection of an aav vector introduced either into skeletal muscle or into the liver in a dog, would result in long-term expression.

we got better levels boy going into liver because liver is better at secreting a gene product directly into the circulation. but you can see that these animals that were dosed at times zero were still expressing therapeutic levels, 140 weeks

later. we followed some of these animals for as long as a decade and it's a very long lasting effect from a single infusion of vector into the portal vain or the hepatic artery. and similarly, when we did studies in humans where we

introduced an aav two vector into skeletal muscle in a man with severe hemophilia, we recently had the opportunity to go back and sample muscle in a patient who had been injected with aav2 factor 9 ten years earlier and there was still factor 9 being expressed in the

injected muscle. this is histochemical staining for factor 9. however, in the first subject in a liver trial to receive a therapeutic dose, instead of seeing long term expression as we had in the hemephilic dog lever and in humans skeletal

muscle, instead, we saw diagrams here in red expression at a therapeutic level in this patient for about four weeks and then he slowliy lost factor 9 expression until he returned to his baseline of less than 1% this is a series of different animals models time course we

hasn't seen. it was a transaminant elevation that occurred in tandem with the loss of factor 9 expression. so, the time course of thissing ised immune meiated response - agod. and subsequently we were able to develop reagents to look at this

specifically. this is using a pant merto examine the kas id specific cd8 t-cells in a subsequent patient who was infused with aav factor 9. you can see what happened in this patient, he didn't get detectable factor 9 expression

at all but he did experience this transamnas elevation and when we used this pant merto look at the population of capsid-specific cd8 t-cells, we could see that population of cells expands after vector injection, and then it contracts n a time course that matches the

rise and fall and in liver enzymes and other experiments we were able to show that the specific epitope that was recognized by the patient's cd8 t-cells was highly conserved in a number of different aav capsids and that they evoked the same response when the patient's

lymphocytes were exposed to the appropriate epitope. so, there was cross reactivity with a number of stereotypes for this particular epitope. so, our working hypotheses shown here that the caps sid essentially a preformed antigen, that after the vector enters the

cell in the end zome and escapes from the end zome, it eventually uncodes and the dna goes to the nucleus and begins making factor 9 and some of the capsid is left behind in the cytosol and we know based on work that was done by john engel heart and others, that that capsid left behind

under those protostomeal processing, and can eventually be transported into the er where it gets loaded on to class one, displayed on the surface of the transduced he pat sight and makes that cell a target for circulating cd8 t-cells, which, because humans have memory to

aav where others species do not, results in the destruction of the transduced cell. and based on other experiments, we have been able to demonstrate the level of antigen presentation is dose-dependent so the higher the dose that is delivered into the cell, the

greater the likelihood that these transduced hepatocyte will be detected and destroyed. so a number of other hypothesis were proposed to explain that series of findings. and one was that the vector had during manufacture, packaged the rep/cap plasmid so the patient

was continuously expressing capsid. and predicted that short-term immunosuppression wouldn't work. another pie pot sis there were alternate open reading frames in our -- hypotheses -- and that the immune response was directed towards those.

and there were predictions that if we switched serotypes we would avoid the response because of aav2 serotype transduced antigen presenting cells more efficiently than some other serotypes. our hypotheses about pre-formed capsid led to the prediction

that short-term immunosuppression would work as an approach if one could simply block the immune response until the pre-formed capsid of the degraded in the cell. one could perhaps see long-term we did a series of experiments to try to support that

hypotheses and i'll just point out maybe a couple of those. we eventually cloned the t-cell respentor from a capsid-specific cd8 t-cell and then you're able to use this as a detecting reagented to detect peptide mhc complexes carrying peptides derived from the aav capsid.

this is some of the date a told you we had that suggested this immune response was dose dependent. if you take that t-cell receptor and clone it into a t-cell line that expresses luciferase in proportion to the degree that it recognizes its cognate peptide

mhc complex, then, if you take human hepatocyte transduce them with aav capsid, you can show that at increasing levels of capsid multiplicity of infection of the vector, you get progressively increasing expression of luciferase. so it demonstrates that the

amount of antigen displayed on the surface of the transduced cell is dose dependent. so the other important thing that we learned in that initial trial and the work they told you about was done over years after that first trial, but the other important thing we learned in

that first trial, these two patients both got the same high-dose of that first vector. and one of them had detectable expression for a period of weeks until the cd8 t-cell response super vened, whereas the other one, got very little deductible expression and four weeks was

down to zero. this person had pre-treatment neutralizing antibodies to aav, fairly high tighter, 1-17. whereas this person had a low tighter of neutralizing antibodies. so, the other important thing we learned then was that this

pre-existing humoral immunity to aav would be blocking if we chose to in fuse the vector through the circulation. and we had considerable data in the original muscle trial that said that if you're going through skeltal muscle, these antibodies aren't a problem.

clearly if you're going through the circulation, they would have ample opportunity to neutralize the infused vector. this is from a study done by jim wilson. these are percent of the population with neutralizing antibody titers of greater than

1-20 and the percentage of the population fairly substantial across four continents. and that is 1-20 fully blocking. so, really we are interested in the population with even lower neutralizing antibody titers. so the second aav factor 9 trial was conducted at university

college london and st. jude children's research hospital. so the solution they proposed to those two problems with the immune response that had been seen in that first trial were shown here. first just exclude everybody with neutralizing antibodies.

so that's the long term solution. because it involves too many adults and there are a lot of adults with hemophilia. but it's a good short-term and then, they added a provision that a short course of steroids would be administered if the

liver enzymes rose or the factor 9 level began to decline. so, in addition to that, they did a number of things to make their vector more efficient. they used a serotype that has a strong tropism for liver. they used a soft complementary vector design so normally they

are single stranded. theirs was double-stranded so it didn't have to rely on the host cell machinery for second strand synthesis and it was codon optimized. so the goal and the expectation was that it would be possible to in fuse a lower dose and still

get therapeutic levels of expression and perhaps at the lower dose, the t-cell response wouldn't be encountered at all. and in fact in the first subject it was infused in that study, this appeared to be the case. this patient was infused now over three years ago and he

after infusion of two times 10 to the 11 vector genomes per kilogram, his circumstance latting factor 9 level went from less than 1% up to 2%. that's a low-level it's modest. but it is definitely different from less than 1%. this patient has been able to

reduce very substantially the amount of clotting fabber he uses to manage his disease. so for him, 2% really mostly removed the need for prophylactic factor 9 infusions. however, as dose escalation continued, what was seen at this dose, two times 10 to the 12,

initially the patient had a circulating level of 6-8%, which is great, that really converts severe hemophilia to mild. but starting at about seven weeks after vector infusion, the liver function tests which had been completely normal, began to rise and then they rose very

quickly over the period of a few days. and that was of course the pre-determined trigger for initiating an eight-week course of prednisolone. and this rapidly renormalized the liver function test and stabilized the factor 9 level

and eventual at the stabilized between 1% and 2%. so, we have the opportunity to study the lymphocytes from this individual using interferon gamma elispot and you can see the time course tracks closely to what happened to the liver function test.

you begin to see some modest level of response above the baseline at 5-6 weeks. and then, at eight weeks it shoots up to a markedly positive response in parallel with this rise in the liver enzymes. and then after the initiation of prednisolone, these cells

rapidly disappear from the so, the clinical investigator in london, ted tud numb elearned a great deal from that. if you look here at the frequency with which he checked the liver function test, after that previous patient, you can see he was extremely vigilant.

and as soon as he started to go up at all, he started the patient on prednisolone and that patient has stabilized now two years later with a circulating level of around 5%. again, what we saw here was a very modest response initially and then it shoots up at 9 weeks

just at the point where it appears that the lfts are starting to rise each very modestly. so, since then, they treated several more patients. and this was presented at ash and you can see most of these people are stabilized in a range

of 4-6% now and so, that's very exciting results. four of the six people needed that course of steroids. two of them went through with never raising the lfts at all so one of the important goals for the field is to try to determine what percentage of

adults are actually going to need this course of steroids. how many people will have this response? and of course another way to look at it is, the easy way to do it is just if you know exactly when this is going to happen, can you just give four

weeks ever steroids and not worry about tracking it? and so i think what is important about that high dose cohort is impossible to identify dose that leads to long term expression at therapeutic levels. the transamnas elevation is asymptomatic but serves as a

marker of the cellular immune response and triggers the investigator to start the course of steroids which seems to stable ice the transduced hepatocytes even though administered for four-8 weeks. for most hemophilia patients, the risk at least short-term, of

this series of events would far outway or be far outweighed by the benefit of long term expression of a level of factor 9 and the range of a few percents. so what i would say about where the field is now in developing av mediated gene transfer to

liver, we understand much better the problems posed by the humoral immune response and cellular immune response and we believe the doses shown here two times 10 to the 12 per kilogram is possible to control the cellular immune response way short course of steroids.

and so i think it will only be answered and continuing to follow large animals and human subjects who have been infused with vector and so, obviously all of these patients are followed for life. so let me just say a word about what we have been trying to look

at as a strategy for the 40% of the adult population with pre-existingents to aav. so, we got our idea from this by looking at comparing the results to the first two trials. the aav2 factor 9 trial, which had a single stranded dna vector, and the av8 trial which

was self complementary, but in the end the doses in these two trials ended up being about the same. and at the high dose cohort, at least before the t-cell response super vened, you can see that both of them led to a similar circulating level of factor 9

which suggests that the potency was similar. however, in the av2 trial, there was no expression at the low-dose cohorts whereas in the av8 trial, there was expression, at least a modest level at the lower to medium dose cohort. what was the difference?

what accountedded for this? was it the stereotype? the difference in the dna confirmation? we wondered about another difference between these two preps. the prep administered in the ucl st. jude trial had a ratio of

about 10-1 empties to fulls. whereas the production process that was used to make that vector, which was made at afa jen, separated the fulls from the empties. and so, what was infused into the two subjects differed in that way.

and so, we wondered whether the empty capsids might in fact serve as decoys to absorb circulating antibodies to aav resulting in better transduction efficiency particularly at the lower doses. so, we carried out a series of experiments done by another lab,

and they used a mouse model. the mouse was injected with human ivig at a range of doses to mimic neutralizing antibody titers encountered in the human population and then the mouse was injected with aav factor 9 vector with no empty capsid or with a 10 fold excess of

empties. here is what you see. if the mouse has no neutralizing antibody tighter, it doesn't matter whether you add empties at 10 fold excess or you don't, you get the same level of factor 9 expression. if you give the mouse a low

tighter neutralizing antibody to aav, then if you don't add any empties, you don't get much expression and if you add a 10 fold access of empties, you get very nice levels of expression. and so, fredrico went on to do a series of experiments where he just gave mice progressively

higher neutralizing antibody titers using ivig and then showed that by adding progressively higher levels of empties to the final formulation that you could restore levels up to that seen in a naive mouse but the higher the antibody tighter, the higher excess of

empties is required to achieve that effect. and if the mouse has a very high neutralizing antibody, you can not overcome it because you start to inhibitry ceptor mediated uptake with the vast excess of empties. so, the way to make this even

safer of course is to alter the receptor binding sites so the empties, when they are used, can circulate but they can never gain access to the target cell and contribute to the burden of capsid that will be processed and presented on the surface of the transduced hepatocytes.

so that is another strategy that we are interested in. andy woo think that eventually for use of this kind of strategy in people with hemophilia, we will have a sensitive assay with neutralizing antibodies to a personnelides final formulation of the product.

so, one of the -- so we are testing this and in our ongoing trial, at children's hospital. and so, in that trial, we will be using this same expression that we had used in the first aav2 trial but in an aav8 and then the final formulation will be linked to the patients

neutralizing antibody tighter. so this trial is open and ongoing at children's hospital and university of pittsburgh now. so, i think for the field as a whole around hemophilia, obviously there are a number of very important questions.

what will be the duration of expression in the ucl accept jude trial? there is now over three years of expression in the first subjects with observation ongoing. will it be possible to readminister and what will be the role of neutralizing

antibodies which always rise after vector infusion? will it be possible to pursue the same approach for hemophilia a and certainly for protein therapeutics, there is a much greater risk of immunogeisity in factor a deficiency compared to factor 9 deficiency.

and then of course, the long term related side effects of this type of therapy? we have followed over 77 dogs with severe hemophilia, a and b, for periods of over 10 years, and have not seen long term side effects related to this therapy. and then the subjects are on the

first trial were all in fused between 2001 and 4, they are part of a long term follow-up study with no evidence of problems. and will it be possible to extend this to the pediatric population? so, what i'm going to do, i see

that as usual, i have been overly ambitious about what i can cover. what i would like to do, though, in the remaining minutes, is turn to a situation. this is an application that we have been working with over the last 7 years with jean bennett

and albert mcguire at the department of opthalmology at penn. if you spend a lot of time worrying about immunely responses to your vector and transgene product, it's always tempt to think about a target tissue that is relatively

immunoprivileged. and of course, that is what the subretinal space represents. and in a gene that -- and jean worked for a number of years on a particular genetic form of inherited blindness congenital amaurosis. but it is an autosomal recessive

defect and all leber's is characterized by the early onset of retinal degeneration and these children are diagnosed in in fancy or early childhood when the parents notice the baby does not track visually. 10-20% of the cases are due to mutations in rpe65 gene.

and there is a naturally occurring doing model. -- dog model. this is a list of genes that if they contain mutations, can lead to lca and as some modest proportion of all of these cases are due to mutations in rpe65. so from an anatomic standpoint,

the mutation effects the retinal pigment epithelial cells where rpe encodes isomberaise. so when like strikes the retina, you get this isomberrization reaction that then starts an action potential that goes back to the optic nerve. eventually this transretinal is

transported back out of the photoreceptor into the rpe cells where it must undergo this isomberrization to generate this again. if you do not have the activity of this enzyme then the visual cycle is broken and blindness results -- could restore vision

in these animals as long as the animal is treated relatively early in life and the reason for that is that as the rpe cells -- that full function of the rpe cells is reel -- really required for the photoreceptors to survive. and that eventually these will

degenerate and then it's not possible to restore vision even if the enzyme is re-introduced. and she and her colleagues were able to flush that out a little more clearly in a mouse model. so let me just say among the many things we did here, ready for this trial were device

studies and this is one of the devices approved for subretinal injection in the united states. and one of the points we learn is illustrateed here if you don't put a small amount of ser fact in into your final formation, the low concentrations used in this

setting result in most of the materials sticking to the delivery device and the delivery is less than was initially loaded into the syringe. so we do put a small amount of surfact in into our final so after completion of a series of studies to optimize the

vector and to decide on the final formulation, this trial was initiated at children's hospital. we initially limited -- limited it to children at least 8 years old but young adults not more than 27, because of that dog data.

and the dose escalation design included three subjects in each of three dose cohorts. because children were going to be included in the trial, the irb insisted - each the first dose we use was one that showed efficacy in more than 90% of the affected dogs and the reason for

that is that if children are involved in clinical trials, if it involves more than minimal risk, which this sort of injection does, there must be the prospect of direct benefit for that child. so we had to start a new dose that seemed to work in dogs.

so, after extensive baseline testing, subjects are enrolled in the study and is injected. the first subjects were enrolled in the fall of 2007 and this shows the data on the first 12 people who were in the phase i-two study. they now have been followed for

a period of over the longest part, over 5 years we collected data on visual acuity, visual fields and light response and mobility as a that was set up in the opthalmology clinic that has a series of obstacles the patients has to step over and around to get to a door at the

end and the results for the injection of the first eye showed that for 9-10 subjects the injected eye develops a stronger poop larry light reflex and no uninjecteddize developed a stronger plr. increased light sensitivity into the injected eye and that didn't

happen for any of the subjects in the uninjected eye. 4 of 8 subjects demonstrate the improved navigational ability using the injected eye but nobody did that for the uninjected eye and similarly, 50% of the patients showed improvement in visual acuity,

more than 3 lines on the eye chart in the injected eye whereas nobody did that in the uninjected eye. so, if you never seen these videos of children going through the mobility assay, they are very impressive. i'll show just a minute or two.

what you will see in this first video is three months after vector injection into the worse eye. the subject first has his injected eye patched and he is trying to go through the mobility course using his uninjected eye, which was he

wasly his better eye. and what you see, he is supposed to be following a black arrow on the floor and even at the very beginning, he wanders off the course. and that is embedded in the background redirecting him. and i think you don't have to

watch it for very long to understand that this is really blind behavior. and then when the patch is applied to the uninjected eye and the patient is asked to go through the mobility course, using his injected eye, you can see a very marked difference in

his performance in these two situations. so, to move this forward, the fda i was interested in knowing the answer to whether it was safe to inject the other eye. and so, we initiated that study in november of 2010. we had to write in the consent

form for all of these people that it was possible that they would develop immune response that would wipe out the gains in the first eye. and so, fortunately for us, three of the adults were willing to sign that consent form and to undergo the injection.

there was an 8 week stagger between subjects to try to monitor for any evidence of immune response, which did not occur. and because it appeared safe in the first three subjects it was possible to do the repainer of the individuals except for one

individual who had glaucoma in the cont lateral eye. so he couldn't undergo injection of the second eye. currently, we are engaged in a phase iii study of this and i think that you can understand that one of the controversial issues is what to use as the

control. the fda was concerned that if we used one eye injected and the other eye is control, this controls for any affects due to the specific mutation that the patient has, all of these people have different mutations in their rp65 gene but of course

the product will not, in the end, be administered to one eye. it will be administered bilaterally. in the end, what we decided to use was subjects that they are deemed eligible, if they meet all the eligibility criteria, they are randomized 2-1 either

to be injected bilateral, nonsimultaneous injection within one week, or they go into a control group and the control group undergoes all the same testing, but one year later is permitted to crossover to the treatment group. this study is being conducted

both at children's hospital and at the university of iowa and enrollment is proceeding very nicely and we hope to have finished enrolling that study by early 2014. so, i think what i'm going to do now is just unfortunately, i'm not go to have time to talk

about the work we have been doing with genome editing. very interesting approach but let me just summarize and say that in vivo gene transforwith the av vect source a clinical reality. it's a licensed clinical product in europe.

recombinant aav vectors are not viruses. but these encounter or evoke immune responses in humans and it's important to try to understand those to characterize them so that they can be appropriately managed. but for those of you who do

allogeneic bone marrow transplantation, understanding and managing immune responses with aav vectors is way easier than transplanting bone marrow. and each target tissue or route of administration represents a unique problem in terms of immune responses.

we have a very nice platform for delivery to the subretinal space and to the liver at the doses that have been used so far and that that provides much scope for further development in the field. so i want to just say that in our group at the children's

hospital, the work on immune responses has been led by fredrico mingozzi who has taken a faculty position at genathon and also by these others. read read. [ reading ] our collaborator for all of the studies in hemophilic dogs has

been tim nicholls at u in. c-chapel hill. that jean bennett,al mcguire and others have been our partners in the lca2 trial and a number of other investigators at children's hospital involved either with clinical grade vector production or with

preclinical and clinical studies in hemophilia and greg with lca2. our collaborator on many of our immunology studies gunnedy ertl. man collaborators from stan formed, af i jen, sidney and brazil. and the aav8 factor trial

conducted by. [ indiscernible ] thank you very much for your attention. and i'm happy to take any questions if there is time.