| Disclaimer: The information contained within the Grand Rounds Archive is intended for use by doctors and other health care professionals. These documents were prepared by resident physicians for presentation and discussion at a conference held at Baylor College of Medicine in Houston, Texas. No guarantees are made with respect to accuracy or timeliness of this material. This material should not be used as a basis for treatment decisions, and is not a substitute for professional consultation and/or peer-reviewed medical literature. Cochlear Gene Therapy: Where We Are and Where We Are Going Today’s topic is a hot topic of research in several areas of the country: cochlear gene therapy. I would like to just give an overview of this subject as well as what is going on here at Baylor. The patient I would like to present is a three-year-old white female with bilateral profound sensorineural hearing loss. She was a product of a normal, uncomplicated pregnancy and delivery and, other than mild newborn jaundice, her neonatal period was unremarkable. Her past medical history is negative. She has no history of developmental delay, and her physical examination was normal as well. A CT and MRI of the temporal bones was obtained at the age of 10 months, which werewithin normal limits. Her TORCH titers and FTABS tests were negative. She does have an older sister with less severe hearing loss; however, her family history is otherwise unremarkable. The patient babbled at six months of age but otherwise developed no intelligible speech. At 10 months of age audiologic examination demonstrated no response to air- or bone-conducted clicks or air-conducted tone bursts in either ear at equipment limits. She had Type A tympanograms bilaterally, and DNA testing revealed compound heterozygosity for mutations in Connexin-26. The patient was tried with hearing aids for approximately six months but gained little benefit from this, and so cochlear implantation was performed at age 16 months. Her speech and language continues to improve since the placement of the implant. The reason I present this patient today is essentially to illustrate that this is the typical patient that will benefit from the technology which is in the ongoing research which we are talking about today. The objectives for today’s talk are to describe gene therapy in general, including the definition and its current uses, as well as the steps and obstacles to success in gene therapy. I would like to discuss the related viral and non-viral vectors, which are available for use to deliver genes to tissues, to discuss why the cochlea is a good candidate for gene therapy, and to describe the various techniques used to deliver genes of interest to the inner ear, as well as their associated difficulties. I would then like to spend a little time on the available animal models we have for this kind of research and to end up by discussing the current and future research in cochlear gene therapy being conducted here at Baylor College of Medicine. In its simplest form, gene therapy can be defined as the delivery and expression of a gene to ameliorate a diseased state. On a deeper level, however, gene therapy can be defined as a targeted introduction and regulated expression of a gene that will produce a therapeutic effect in the desired cell or tissue using a genetically engineered vector to achieve a biological effect. The current uses for gene therapy in clinical trials around the world include mainly cancer, infectious diseases, and single gene defects such as cystic fibrosis, hemophilia, severe combined immunodeficiency, and muscular dystrophy just to name a very few. There is actually some very exciting work going on here at Baylor College of Medicine by Dr. Brian Butler, in Radiation Therapy, in his research with prostate cancer using gene therapy combined with radiation therapy. He is actually getting very exciting results. The first clinical trial for gene therapy began in 1990, and currently there are over 500 clinical trials underway worldwide. However, the initial enthusiasm for this technology has given way to guarded skepticism because of only isolated sporadic reports of clinical success. So what are the steps to success when deciding to perform gene therapy? First, you must determine what your desired biological effect is, and there are several questions you must ask yourself. Is this going to be a localized toxic effect on a tumor cell? Would you like to replace a missing gene product or produce a therapeutic protein or introduce an inhibitory gene to your tissue of interest? Once this is decided, you then must determine what your target tissue of interest is. Questions to ask here are: How accessible is that tissue? Will the gene of interest be introduced directly in-vivo or will ex-vivo transfected or transduced cells be introduced to the site of the target tissue? There are also several roadblocks, which are occasionally stumbled upon when performing research in gene therapy. The first is with problems with the gene delivery system. The gene must be targeted to the correct cell population, and this can be modified in one of two ways. You can either inject the gene locally to the tissue of interest, or you may choose a vector which targets the selected cell type. Also, the lack of sustained expression of genetic material has been a problem in the past, as well as the host immune response to foreign material with several of the viral vectors, as we will see in just a few minutes. When talking about how to deliver a gene to the tissue of interest, the two main categories are either non-viral or viral with several examples, which we will go into more detail about. The first of the non-viral means is called “electroporation” and what it is, is essentially cells in tissue culture are subjected to an electric current which induces transient cell membrane permeability. The gene of interest, which is then contained within a plasma, is able to enter the cell and express its effect. The advantages of this method are that it does not require the use of a second agent such as a lipid or virus; therefore, there is no immunogenicity or toxicity associated with it. However, it has a very poor efficiency of DNA transfer, and there is an increased risk for permanently damaging the cells with the electric current which they are subjected to. The second of the non-viral vectors are cationically charged liposomes. They are basically DNA-containing lipids within a bilayer lipid membrane, as seen in the picture. The advantages of this are that there is no size limitation to the inserted gene, there is no immunogenic effect to the host, they are very easy to produce in the laboratory, and there is no risk of viral or vector-related disease or toxicity. However, again they have a very poor efficiency of DNA transfer, and there is almost no control over the tissue targeting other than where you inject the vector itself. The most common means of transferring genes to tissues is by using the natural infectivity of viruses. These viruses have been highly modified in the laboratory to be replication deficient and, just for a basic review, the structure of a virus is that of a capsid or protein coat which within it contains either DNA or RNA which can be either single or double stranded and, as we all know, viruses infect host cells by a number of different mechanisms and replicate their genetic material within that cell of interest. Among the earliest viral vectors developed were the retroviral vectors. They have been used in many research studies for gene therapy. They contain a single stranded RNA genome, which encodes for three main genes: the core proteins, the reverse transcriptase, and the coat proteins. Once the RNA enters the cell it undergoes reverse transcription, and the viral DNA then integrates into the host cell genome. Because it integrates into the host cell chromosome, this provides for stable, long-term expression of the gene of interest. However, the main problem with retroviral vectors is that the DNA insertion into the host cell chromosome is a random event. Therefore, it places that cell at a risk for insertional mutagenesis. They also only are able to infect dividing cells, which makes them very useful for the rapidly proliferating cells in cancer. However, they would not be suitable for the post-mitotic neurosensory epithelium of the inner ear. Lentivirus is the next category. It is actually also a retrovirus, which is based on HIV. However, what makes it different is that it integrates into the chromosome of dividing and non-dividing cells, which again provides for potentially long-term expression of the gene of interest. Herpes virus vectors are double stranded DNA viruses with a tropism for neural tissue, in-vivo and in-vitro. They can enter a latent state in some neuronal cells, which again provides for potential long-term stable gene expression; however, this is usually limited to only a very few cells ,and it is difficult to control what cell it becomes in its latent state. The disadvantages of using herpes virus vectors are that they are difficult to produce in the laboratory, and they have a low infection efficiency. Adeno-associated viruses are vectors which have gotten a lot of press lately in the gene therapy literature. They are small, single-stranded DNA parvovirus, and are endogenous to many mammalian species. They are not known to cause any human disease; therefore, it makes them more attractive for vector delivery. They are able to infect non-dividing cells, which could potentially make them useful in the cochlea, and there is usually a second helper virus which is usually adenovirus needed for replication of the virus. It does integrate into the host cell DNA, which again provides for long-term stable integration of the inserted genes; however, the disadvantage is that it is a very, very small virus and the size of the gene, which is inserted into, it is very limited. Also a very popular vector in gene therapy research and what we are using in Dr. Oghalai’s lab is the adenoviral vector. It is a linear double stranded DNA virus, and it is a common human pathogen causing colds and conjunctivitis. However, the replication of defective vectors, which are produced in the laboratory, have been deemed safe in several studies. Adenovirus is able to infect dividing and non-dividing cells; however, it does not integrate into the host cell genome. This has the disadvantage of only having short-term expression of the gene on the order of three to six weeks in most cases. The other big disadvantage of the adenovirus is they provoke a strong immune response in the host, which may be toxic to the target cell. However, there is new vector development going on right now which focuses on the elimination of excess viral genes to minimize the host immune response; and these viruses, which are basically devoid of all the viral genome and only have the gene of interest in them, are known as either gutted adenovirus vectors or helper dependent adenovirus vectors. And, as we will see in a few minutes, this is actually what we are using in our lab. The advantages of adenovirus are that they are capable of infecting a wide variety of non-dividing cells, they are very easy to produce in the lab relative to other viruses, and that makes for availability of high titers for use in gene therapy. They also have a very high transduction efficiency. So what makes the cochlea a good candidate for gene therapy? Well, first of all, its being encased within the temporal bone; the cochlea is relatively isolated from other organs. This allows for a localized injection of the viral vector containing the gene of interest to the tissues of the inner ear. Also, a high concentration of the drug is able to be applied without affecting other tissues for the most part. The cochlea for the most part again is a closed system, although the cochlear aqueduct can be patent in some cases, potentially providing a route to spread to the CSF. What I mean by a closed system is that the perilymph and endolymph of the cochlea is able to disseminate particles, which is injected within it very quickly to reach the entire cochlea. Also, the cochlea has several very well known cell types, which may be precisely quantified by microscopic and other means. And there have been a number of studies, as illustrated here, which have tested several different viral vectors and non-viral vectors, including cationic liposomes, adeno-associated virus, adenovirus, rhinovirus, and herpes simplex virus; and they have studied their efficacy utility and safety, and the general consensus is that they are efficient and they are generally safe to use. So once you have chosen your tissue of interest and you have chosen your vector to deliver your gene, you must then decide how are you going to get it to the tissue of interest, the cochlea in this case. The goals of vector delivery method in the cochlea are to preserve hearing function and to preserve cochlea architecture. One way to do this is to administer the vector systemically; however, this has the drawback of systemic toxicity. So most have chosen direct instillation to the cochlea, which you are able to deliver a high vector concentration, it increases the likelihood of gene delivery to the tissue, and it minimizes leakage of the vector into non-target organs. So how do we do this in animals? Well, the first method, which has been described, is by using an osmotic mini-pump to infuse or to inject the vector into the round window membrane directly through the round window into the scala tympanum. The second, which is illustrated here, is by drilling a cochleostomy into the basal turn of the cochlea and again using an osmotic mini-pump or microinjection techniques to deliver the vector that way. It has also been described to inject it into the endolymphatic sac, which is accessed from the posterior cranial fossa, as well as infusion into the posterior semicircular canal. And most recently in 2001, out of UCSF, they placed a piece of gel foam which had been soaked with the viral vector solution and placed it on the round window membrane and were able to prove that adenovirus and liposomes, but not adeno-associated virus, were able to diffuse across the round window membrane and, in fact, infect most of the supporting cells not actually the inner and outer hair cells, mainly in the basal turn of the cochlea. All of these procedures are able to be done; however, they are very difficult because we are doing it on very small animals and it is very difficult to accomplish our goal of vector delivery, which is preservation of hearing and preservation of cochlear architecture. So historically in gene therapy, inner ear gene therapy practical considerations have factored into the selection of the animal model, including the size of the animal, the anatomy of the external middle and inner ear, and also the ease of surgical access to the cochlea. What has come out of this is that the guinea pig has been the favored species for many inner ear studies. And until recently, the mouse has been precluded as a model secondary to its small size and technical considerations. The mouse cochlea is only one-third the size of the guinea pig, and another element of difficulty is added when you take into consideration the persistent overlying stapedial artery, which makes manipulation of the cochlea very difficult. However, despite these surgical difficulties, there are several advantages to using the mouse in this study. Mouse genome is being rapidly sequenced. Their generation time is relatively short. Their mutations are very easy to trace, and there are also significant developmental, anatomic, and functional similarities between the mouse and the human ear. Perhaps the biggest advantage is that there are already several hereditary inner ear diseases, which have been matched with homologous mouse models. This chart shows the known human diseases which cause hearing loss, as well as the mouse model associated with that disease; and very recently in the last year or so, the Connexin-26 mouse model has been able to be produced. They had a problem for a long time with the mouse actually dying; the mutation was lethal in those mice, but it is available for use now. So what are we doing in the laboratory of Cochlear Mechanics and Hearing Loss headed by Dr. Oghalai here at Baylor College of Medicine? We came up with the question that mouse outer hair cells are able to be infected by helper-dependent adenovirus vector. What makes this study different from others is that we are actually using a mouse with all of its technical difficulties, and we are also using a helper-dependent adenovirus vector which, as we will see on the next slide, is different than a regular adenovirus and has not been used in the cochlea to date. Helper-dependent adenovirus is devoid of virtually all viral DNA. Therefore, it cannot replicate by itself and it needs help from a helper virus. The foreign gene is inserted into the viral vector surrounded by stuffer sequences, which take the place of the viral DNA. There is a packaging sequence here, which allows it to be packaged within the viral capsid. The helper virus also has this packaging sequence and the helper virus has all of the genes, which are necessary for production of the viral capsid and packaging and other things. These two viral vectors are put into tissue culture and grown and, through sophisticated means, the packaging sequence of the helper virus is cleaved off, not allowing that helper virus to be packaged, and only our vector of interest is packaged. This is then reamplified in the tissue culture until sufficiently high titers of vector are produced. This actual virus is produced by Dr. Ng in the Department of Molecular and Human Genetics at Baylor, and his research is with mainly liver. These pictures prove that this virus works in baboon hepatocytes with low concentration of vector and high dose helper-dependent adenovirus. As you can see, virtually all of the liver cells have been transfected with LacZ as an indicator. Wherever blue is, the virus is. So in our lab, we first determined that we could perform this technology actually transfecting adenovirus culture into cells. We first infected HEK cells with a regular adenovirus vector which was labeled with GFP as our indicator protein. We were successful in transvecting HEK cells, and then we performed immunohistochemistry with an anti-GFP antibody to precisely determine where the GFP was in those cells. After it was clear that we could do this, we then decided to inject it into the mouse. The way you access a mouse cochlea is to make a paramedian incision usually on the right. You dissect down into the tissues—I am sorry about the quality of this picture—but this actually shows the mouse temporal bone and the bulla is actually just one bulla; once it is entered you have access to the whole middle ear. So this picture shows, after the bulla has been opened, this is the medial edge of the tympanic annulus with the tympanic membrane right here, and you are able to see the cochlea in the floor of the temporal bone. Your useful anatomic landmarks for identification of the cochlea and where you need to inject the virus are the persistent stapedial artery, which runs right over the round window, which can be easily seen here. So once we identify our anatomy, we then use a 1 mm micro-drill to very carefully drill a cochleostomy in the basal turn of the cochlea just above the stapedial artery. We drill away just enough bone to expose the lateral wall of the spiral ligament. We then take a glass-tip pipette, which is very difficult to see, but this is the tip of the glass pipette which has the vector solution already in it. We then inject this solution into the scala media through the spiral ligament. We used a concentration of 5 times 10 to the 10 th viral particles per milliliter at a rate of 1 microliter per minute of vector. The mice were then sacrificed on post-operative day #4, and their cochleas were harvested from the temporal bone. They were then stained for LacZ which is our indicator to determine where the adenovirus was, and which cells were infected. LacZ is actually a bacterial enzyme, which cleaves glucose and the lactose in galactose and, when reacted with X-gal, produces an intense blue dye. So, once we did this, the otic capsule was then removed very carefully, and the organ of Corti was micro-dissected and placed in a whole mount prep so that we could examine the cells of interest. And so these are just pictures of our control cochlea, which had no virus injected, and then the injected cochlea, and you can easily see the cochleostomy here. The virus was injected into that cochleostomy, and you can see that there is blue staining within that cochlea which appears to be along the basilar membrane in a spiral fashion. So we then carefully picked away the bone of the otic capsule, and this is a picture of the modiolus, the bony modiolus in kind of an oblique axis with the first spiral of the basilar membrane and organ of Corti around it, and it can be seen that there is a definite linear pattern of virus infection. And this is a higher magnification view. So we then wanted to find out which cells were actually infected, and to do this we had to use micro-scissors to carefully dissect off a piece of the organ of Corti and basilar membrane only and to place it in a petri dish. This is after that has been done with a control cochlea and the injected specimen. And as you can see, compared to the control, there is a lot of blue staining which would suggest that the virus was able to infect some cells in here, including the neurosensory epithelia of the inner ear which we will see at a higher magnification view next. This would be the spiral ganglion cells right here. So this is at a 40-power magnification and in the control specimen, you can easily see your first row of inner hair cells right here separated from the three rows of outer hair cells by the pillar cells in tunnel of Corti. And as you can see, there is a definite linear pattern of staining within the injected specimen along all aspects and all cells of the scala media. You can see a definite row of inner hair cell stereo cilia right there, as well as at least one row of outer hair cells here and possibly a second row here. It is very difficult at this stage to tell exactly what cells were infected, possibly the outer hair cells were infected, but it could be underlying cells since this is a whole mount tissue prep. So what we may do in the future is, before we do this whole mount prep, we may embed the cochleas in paraffin and then slice them into thin sections and perform immunohisto-chemistry to precisely determine what cells are infected. So, the conclusions that we reached were that helper-dependent adenovirus vector does appear to infect cells of the inner ear, possibly including the hair cells, when injected into the scala media of the mouse cochlea. And as I have stated, we need to more precisely determine which cells are infected in the future, as well as to determine the hearing preservation in the mice subjected to a cochleostomy and scala media injection of helper-dependent adenovirus vector. This will be done through ABRs and distortion product otoacoustic emissions, which we have the setup to do. Also, we need to determine the toxicity of any of the helper-dependent adenovirus to the outer hair cells as well as the inner hair cells and to determine the optimal concentration and volume of vector which is needed to minimize the toxicity to the neurosensory epithelia of the inner ear. What is very exciting is where we are going with this in Dr. Oghalai’s lab in the future. We are currently underway developing experimental procedures which are delivering the prestin gene contained within the helper dependent adenovirus vector to the inner ear of prestin-deficient mice. And for those who do or do not know, very quickly, prestin is the protein, which is located in the lateral wall of the outer hair cell membrane and it is very well known to be the molecular motor, which is responsible for outer hair cell electromotility. And it is this outer hair cell electromotility, which is the basis of the cochlear amplifier. Through Dr. Fred Pereira at Baylor College of Medicine, we have a depressed and knockout mouse model, and it can be seen here when determining electromotility of outer hair cells. This is the wild type mouse versus the heterozygous prestin and the prestin knockout. And the prestin knockout mouse has zero electromotility, as can be seen in these graphs here. They have a flat line, no electromotility in their hair cells. The outer hair cells are also shorter than those in the wild type mouse and, as can be seen by the ABRs and distortion product otoacoustic emission, the prestin knockout mouse is essentially deaf. We have also recently completed the vector with the prestin gene inserted into it right here. We are using GFP as an indicator, green fluorescent protein, and this vector is currently at the Vector Development Laboratory being packaged into the virus and, hopefully, will be available for our use in the very near future. What is also good is that it has recently been published in the last several months that prestin has been found in a certain percentage of the population to be responsible for a non-syndromic hearing loss and, with the help of Dr. Raye Lynn Alford, we are currently developing the necessary means to test for prestin deficiency in the population at Texas Children’s Hospital. So this is a little bit of what is going on at Baylor, and it is very exciting with its obvious future clinical applications. Case Presentation: The patient has an older sister with less severe hearing loss. Otherwise, the family history is negative for any other history of vision, hearing, neurologic disorders or genetic syndromes. 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