Periodic Reporting for period 4 - iHEAR (Gene therapy of inherited and acquired hearing loss)
Periodo di rendicontazione: 2023-11-01 al 2024-10-31
According to WHO, approximately 430 million people worldwide suffer from disabling hearing loss, with sensorineural hearing loss (SNHL) being the most reported form (>90%). SNHL is associated with a pathological change in structures within the inner ear, and it can be inherited or acquired. Currently, more than 100 genes that have a role in inherited hearing loss have been identified. In addition, several factors can contribute to the development of acquired hearing loss, including the use of certain medications. Ototoxic drugs known to cause permanent damage include, for example, platinum-based chemotherapeutic agents like cisplatin. Cisplatin can be actively taken up into hair cells (HC) and spiral ganglion neurons (SGN) by various proteins on the surface of cells that act as transporters to bring substances into or out of cells. As a consequence, the presence of the ototoxic drug triggers a series of events in the affected cell types.
As the number of people projected to suffer from hearing loss is projected to be almost 2.5 billion by 2050, hearing loss is clearly an important disease that needs to be addressed. Hearing loss can affect all aspects of a person´s life, from learning difficulties in school, impediments to social interactions and problems in the work force. Although there are some medical interventions, such as hearing aids and cochlear implants, these devices do not provide the complete range of normal hearing and not all patients can be treated with these devices.
Therefore, the overall objective of this project is to develop new treatment approaches to improve hearing in hearing loss patients and to prevent hearing loss due to certain medications. To achieve these objectives, we aim to (1) replace defective / non-functional genes with intact functioning genes as a gene therapy approach to treat hearing loss, (2) protect inner ear cells from factors that may damage them and cause them to become non-functional, and (3) use induced pluripotent stem cells to model hearing loss diseases. These main objectives will help us to learn more about the biology of otic cell development and how we can use gene therapy technologies to modify inner ear cells to improve and protect hearing in patients.
As the number of people projected to suffer from hearing loss is projected to be almost 2.5 billion by 2050, hearing loss is clearly an important disease that needs to be addressed. Hearing loss can affect all aspects of a person´s life, from learning difficulties in school, impediments to social interactions and problems in the work force. Although there are some medical interventions, such as hearing aids and cochlear implants, these devices do not provide the complete range of normal hearing and not all patients can be treated with these devices.
Therefore, the overall objective of this project is to develop new treatment approaches to improve hearing in hearing loss patients and to prevent hearing loss due to certain medications. To achieve these objectives, we aim to (1) replace defective / non-functional genes with intact functioning genes as a gene therapy approach to treat hearing loss, (2) protect inner ear cells from factors that may damage them and cause them to become non-functional, and (3) use induced pluripotent stem cells to model hearing loss diseases. These main objectives will help us to learn more about the biology of otic cell development and how we can use gene therapy technologies to modify inner ear cells to improve and protect hearing in patients.
Lentiviral vectors (LV) are one tool that can be used to modify cells in gene therapy approaches. Over one hundred genes are known to play a role in hearing loss and for almost all of these genes, we have generated LV capable of delivering an intact functional copy of the gene to target cells. Several modifications have been tested to identify the most efficient way to produce LV that can deliver their therapeutic gene cargo in an optimal manner, including extent of target cell modification, expression and functionality of the therapeutic gene. Here, we have tested different promoter configurations to drive transgene expression, including cell-specific expression in distinct otic cell types. We have also explored engineering the glycoproteins used to pseudotype LV with the aim to specifically transduce otic cells. Beyond LV, we activeyl work on adeno-associated viral (AAV) vector-mediated delivery strategies, incl. novel engineered AAV vectors with increased specificity for inner ear cell types were identified and further developed for potential clinical translation.
Many patients who are treated for diseases such as cancer suffer from hearing loss as a result of the therapy needed to eliminate their cancer. Therefore, we have designed novel protocols and adapted published protocols for genome editing to allow generation of inner ear cells that are resistant to anti-cancer therapy with the hope to protect the sense of hearing in these patients. Our initial in vitro results are promising, but need to be verified in vivo. Here, it will also be important to test for safety as well as feasibility. We also developed and published a gene therapy approach to protect inner ear cells from ototoxic drugs like cis-platin and aminoglycosides. These data led to discovery of a small molecule that was also effective in protecting inner ear cells from apoptosis.
Induced pluripotent stem cells (iPSC) are a renewable cell source that can be used to generate all different types of cells that make up an organism, e.g. a human. The great ability of iPSC to proliferate coupled with their capacity to differentiate into all different cell types, makes these cells a valuable tool for research as well as potential clinical applications. We have thus far used iPSC to study the transcription factors necessary for development of otic cells, such as inner ear hair cells and spiral ganglion neurons, which are crucial for hearing. We used genome editing to generate a novel reporter cell line for otic cell differentiation from stem/progenitor cell populations.
Many patients who are treated for diseases such as cancer suffer from hearing loss as a result of the therapy needed to eliminate their cancer. Therefore, we have designed novel protocols and adapted published protocols for genome editing to allow generation of inner ear cells that are resistant to anti-cancer therapy with the hope to protect the sense of hearing in these patients. Our initial in vitro results are promising, but need to be verified in vivo. Here, it will also be important to test for safety as well as feasibility. We also developed and published a gene therapy approach to protect inner ear cells from ototoxic drugs like cis-platin and aminoglycosides. These data led to discovery of a small molecule that was also effective in protecting inner ear cells from apoptosis.
Induced pluripotent stem cells (iPSC) are a renewable cell source that can be used to generate all different types of cells that make up an organism, e.g. a human. The great ability of iPSC to proliferate coupled with their capacity to differentiate into all different cell types, makes these cells a valuable tool for research as well as potential clinical applications. We have thus far used iPSC to study the transcription factors necessary for development of otic cells, such as inner ear hair cells and spiral ganglion neurons, which are crucial for hearing. We used genome editing to generate a novel reporter cell line for otic cell differentiation from stem/progenitor cell populations.
The results we achieved for in vivo gene transfer are beyond state of the art in the hearing field, as such successful modification of inner ear cell types has not been previously shown with lentiviral vectors (LV). We showed that different envelope glycoproteins and internal promoters are suitable for LV-based transduction of inner ear cells, a key finding that greatly expands the possibilities to develop gene therapy solutions to treat hearing loss and other inner ear disorders. For example, some genes that play a role in hearing loss are too large to be delivered by other approaches, so the LV system that we have developed, which can even deliver these very large genes, offers the possibility to correct a broader range of defective genes known to have roles in hearing loss.
Beyond potential LV therapeutic approaches, novel capsid-engineered AAV vectors that showed significant differences in vector tropism compared to parental AAV serotype 2 (AAV2) were developed. The best-performing variant had enhanced intracellular processing and uncoating efficiency and effectively transduced spiral ganglion neurons (SGN) across all cochlear turns at low doses. Additionally, it showed therapeutic potential by preventing SGN degeneration in deafened mice through neurotrophic factor overexpression directly in SGN. Directed evolution of AAV was also used to identify additional AAV variants that strongly target cochlea cell types. These AAV variants also show diverse and unique transduction profiles, which means they each have the potential for use as therapeutic vectors to target different cochlea cell types that are affected in hearing loss. Overall, this research advances LV and AAV vector technologies for inner ear gene therapy, showing improved transduction and therapeutic potential for treating sensorineural hearing loss.
Another major advance we achieved was the generation of a tool to better follow inner ear hair cell (HC) development from progenitor cells. Here, we generated an ATOH1-dTomato iPSC reporter line by genome editing combining a Cas9-induced double-strand break and delivery of a homology donor using an adeno-associated virus (AAV) vector. In summary, we have achieved several technological and scientific breakthroughs with high potential to help understand the generation of inner ear HCs, as well as to treat genetic hearing loss and protect from some forms of acquired hearing loss.
Beyond potential LV therapeutic approaches, novel capsid-engineered AAV vectors that showed significant differences in vector tropism compared to parental AAV serotype 2 (AAV2) were developed. The best-performing variant had enhanced intracellular processing and uncoating efficiency and effectively transduced spiral ganglion neurons (SGN) across all cochlear turns at low doses. Additionally, it showed therapeutic potential by preventing SGN degeneration in deafened mice through neurotrophic factor overexpression directly in SGN. Directed evolution of AAV was also used to identify additional AAV variants that strongly target cochlea cell types. These AAV variants also show diverse and unique transduction profiles, which means they each have the potential for use as therapeutic vectors to target different cochlea cell types that are affected in hearing loss. Overall, this research advances LV and AAV vector technologies for inner ear gene therapy, showing improved transduction and therapeutic potential for treating sensorineural hearing loss.
Another major advance we achieved was the generation of a tool to better follow inner ear hair cell (HC) development from progenitor cells. Here, we generated an ATOH1-dTomato iPSC reporter line by genome editing combining a Cas9-induced double-strand break and delivery of a homology donor using an adeno-associated virus (AAV) vector. In summary, we have achieved several technological and scientific breakthroughs with high potential to help understand the generation of inner ear HCs, as well as to treat genetic hearing loss and protect from some forms of acquired hearing loss.