Objective
Goal
To develop new RNA silencing treatment to specifically inhibit a regulatory RNA molecule called HOTAIR which belong to a class of long non-coding RNA. We will develop this treatment to be used as an intraocular injection and as an eyedrop.
Objectives
In this project, we would like to develop a highly specific and robust therapeutic strategy for DR make this approach ready for clinical application. The study has two aims
Objective 1: To develop an siRNA intravitreal injection (injection inside the eye) treatment targeting HOTAIR for DR.
Objective 2: To develop an siRNA eyedrop treatment targeting HOTAIR for DR
(siRNA = silencing RNA,)
Based on our data, we will explore HOTAIR siRNA treatment using two therapeutic approaches (using two potential medicinal compounds)
1) N-Acetylgalactosamine (GalNAC) modified siRNA.
2) a lipid nanoparticle-coated N-Acetylgalactosamine (GalNAC) modified siRNA.
#1 is a chemical modification of the siRNA which facilitate siRNA’s entry in the cell without assistance from any potentially toxic reagents (aka. transfection reagents, which are used to deliver RNA inside the cells).
#2 will use state of the art nanotechnology which will further facilitate the transportation of the modified siRNA molecule (#1) to the back of the eye.
Background Rationale
Diabetes mellitus (DM) continues to wreak havoc across the world. Globally, 700 million are
projected to have DM by 2045. DM is characterized by tissue damage and the development
of diabetic complications. Among these complications, diabetic retinopathy (DR), i.e, affection of nerve tissues in the back of the eye is the leading causes of preventable blindness in the working age population. To prevent and treat this irreversible visual loss from diabetic retinopathy, a safe and specific treatment is essential.
Although there are some treatments available [using an antagonist to a molecule called vascular endothelial growth factor (VEGF)], there are significant limitations associated with such treatments. For example, targeting intraocular VEGF levels require multiple injections using biologics (Avastin, Lucentis, or Eyelea) may result in side effects such as glaucoma (increased pressure in the eye with its consequences), as well as primary or secondary failure of such treatment.
We have identified an innovative and novel therapy to overcome such challenges imposed by existing anti-VEGF treatments. This treatment targets a molecule called HOTAIR (aka. HOX antisense intergenic
RNA). HOTAIR is classified as a long non-coding RNA, which regulates production of multiple molecules causing deleterious effects. We have discovered that the lncRNA HOTAIR is of significant importance in DR, as the blockade of HOTAIR normalizes, along with VEGF, multiple additional molecules of causing eye damage in DR. We have developed a treatment using a small RNA molecule (i.e, a silencing or siRNA) to normalize HOTAIR, which has been successful in animal and is more robust that the current treatment. Results from the first set of pre-clinical studies (already published) indicate that this treatment can potentially be used for the treatment of DR and other ocular conditions (IOVS, 2021,62:20; doi: https://doi.org/10.1167/iovs. 62.3.20). Our recent preliminary study using a chemical modification of this siRNA further showed more robust HOTAIR inhibitory effects in the animals compared to unmodified HOTAIR siRNA with no notable toxicity. Preliminary data also indicate that we will be able to formulate an eyedrop to treat patients with DR. Hence, this novel and innovative project will impact a significant gap in the treatment of DR and will lead to better living for people with type 1 diabetes. Our goal is to bring this treatment to clinics. For this treatment, we had submitted PCT application on in 2021 and entered the national phase in 2023. [PCT application: US Patent Application number: 63/048389, EFS ID39918968, PCT/CA2021050924.
Description of Project
Background:
Globally, 700 million people are projected to have diabetes mellitus (DM) DM by 2045. DM is characterized by tissue damage and the development of diabetic complications. Among these complications, diabetic retinopathy (DR), i.e, affection of nerve tissues in the back of the eye is a leading causes of preventable blindness.. To prevent and treat this irreversible visual loss from diabetic retinopathy, a safe and specific treatment is essential.
Although there are some treatments available [using antagonist to a molecule called vascular endothelial growth factor (VEGF)]. However, limitations of such treatments include multiple eye injections, development of glaucoma (increased eye pressure) and primary or secondary failure.
Our innovative and novel therapy, targeting a molecule HOTAIR (HOX antisense intergenic RNA). overcomes such challenges. HOTAIR, a long non-coding RNA, regulates production of multiple molecules causing deleterious effects. We have discovered that HOTAIR is of significant importance in DR. HOTAIR blockade normalizes, along with VEGF, multiple additional molecules of causing damage in DR. We have developed a treatment using a small RNA molecule (i.e, a silencing or siRNA) to normalize HOTAIR. This has been successful in animal and is more robust that the current treatment. Results from the first set of pre-clinical studies (already published) indicate that this treatment can potentially be used for DR(doi: https://doi.org/10.1167/iovs. 62.3.20). Our additional preliminary study using a chemical modification of this siRNA showed more robust HOTAIR blockade in the animals compared to unmodified HOTAIR siRNA with no toxicity. Preliminary data also indicate that an eyedrop may potentially be used to treat DR.
Goal:
Our goal is to develop new RNA silencing treatment to inhibit long noncoding RNA molecule HOTAIR for DR as an intraocular injection and an eyedrop.
Aim 1: To develop an siRNA intravitreal injection treatment targeting HOTAIR for DR.
Aim 2: To develop an siRNA eyedrop treatment targeting HOTAIR for DR
We will use two approaches.
1) N-Acetylgalactosamine (GalNAC) modified siRNA(chemically modified siRNA, facilitating siRNA’s cellular entry without any other reagents)
2) a lipid nanoparticle-coated N-Acetylgalactosamine(GalNAC) modified siRNA. (a nanotechnology approach, which will further facilitate the transportation of the modified siRNA to the back of the eye).
Experimental plan:
We will use these treatments in human retinal endothelial cell (a major target of glucose in DR) and in rats and mice with type 1 diabetes. Toxicity analyses and analyses of multiple parameters to examine the treatment effects of vivo and in vitro will be performed. We will use both male and female type 1 diabetic (chemically induced models) mice and rats for short-and long duration. We will carry out experiments at multiple level of complexity including biochemical, functional and structural analyses both in vivo and in vitro.
Concluding remarks:
This novel and innovative project will narrow a gap in DR treatment and will lead to better living for people with type 1 diabetes. Development of RNA targeting treatment with potential use as an eye drop for DR is truly innovative a game changer. Following this project, we will seek funds to do similar studies in larger animals. This project aligns with JDRF’s research strategy of better living. Based on the preliminary data, it is highly conceivable that we will be able to formulate an eyedrop to treat patients with DR. Development of an eyedrop for DR will lead to a better treatment with reduced cost and will prevent the suffering from eye injections. The outcomes of this may lead to significant advancements as 1) no RNA-targeting treatment (which is more specific, robust, with less chance of failure) is presently available for DR and 2) there are no eyedrop treatment for DR.
Anticipated Outcome
We will use these treatment approaches using human retinal endothelial cell (a major target of glucose in DR) culture model as well as rats and mice with type 1 diabetes.
Toxicity analyses and analyses of multiple parameters to examine the treatment effects of vivo and in vitro as outlined previously (IOVS, 2021,62:20) will be performed. We will use both male and female type 1 diabetic (chemically induced [Streptozotocin] diabetic models) mice and rats for short-and long duration. Following toxicity analysis and determination of dosage, we will carry out experiments at multiple levels of complexities staring at the biochemical level, leading to functional and structural analyses both in vivo and in vitro.
Development of RNA targeting treatment with potential use as an eye drop for diabetic retinopathy is truly innovative and a game changer. siHOTAIR therapy has IP protection through Western University. Following this project, we will seek funds to do similar studies in larger animals, for which we will seek partnership with pharmaceutical companies or develop a spin-off. This project aligns with JDRF’s research strategy of better living. Based on the preliminary data, it is highly conceivable that we will be able to formulate an eyedrop to treat patients with DR. Development of an eyedrop to treat diabetic retinopathy will further reduce treatment cost,+potentially reduce toxicity and will prevent the suffering from eye injections This approach will help patients not only from a treatment perspective but also will reduce the suffering of regular eye injections. The outcomes of this innovative project could lead to significant advancements as 1) no RNA-targeting treatment (which is more specific, robust, with less chance of failure than conventional treatment) is presently available for DR and 2) there are no eyedrop treatment for DR.
Relevance to T1D
This proposal is based on the hypothesis that a gene therapy targeting long non-coding RNA ( these molecules regulates the production of other molecules) HOTAIR can be used to treat diabetic retinopathy (DR). Furthermore, this treatment can be used both as intravitreal injection and as an eyedrop.
Nearly all patients with type 1 diabetes will develop DR, a leading cause of preventable blindness. DR is caused by damage to the nerve tissue in the back of the eye and ultimately leads to blindness. Several of bad effects of DR is mediated by a molecule called vascular endothelial growth factor (VEGF) Due to VEGF’s critical role, it is the first-line therapy for patients with pathologic changes in the retina (the neuronal tissues in the back of the eye) such as diabetic macular edema (a major cause of vision loss) and proliferative DR (a late stage disease causing blindness). Such treatments use intravitreal injections of anti-VEGF. However, such therapies have limitations, requiring frequent intraocular injections and local or systemic adverse effects such as increased pressure in the eye (glaucoma) as well as primary or secondary failure. Hence, new therapies are urgently needed
Our preliminary data indicate that the proposed therapies are more efficient and longer lasting with lack of any notable toxicity. Preliminary data also indicate that we will be able to formulate an eyedrop to treat Type 1 diabetic patients with DR. Development of an eyedrop for DR will lead to a better treatment of Type 1 diabetic patients with reduced cost with potentially reduced toxicity and will prevent the suffering from eye injections. This project aligns with JDRF’s research strategy of better living. The outcomes of this project may be a game changer and may lead to significant advancements as 1) no RNA-targeting treatment (which is more specific, robust, with less chance of failure) is presently available for DR and 2) there are no eyedrop treatment for DR. Hence, this project will impact a significant gap in the treatment of DR and will lead to better living for people with type 1 diabetes.