Patent Application
20210253999
N.A.
The present disclosure relates to recombinant nervous system cells and methods to generate them. More specifically, the present disclosure is concerned with recombinant nervous system cells (e.g., cone photoreceptors) and methods to generate them from neuroepithelial cells and adult glial cells.
Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named 2489-PCT-SEQUENCE LISTING-12810-690_ST25, that was created on Nov. 4, 2019 and having a size of 408 kilobytes. The content of the aforementioned file named 2489-PCT-SEQUENCE LISTING-12810-690_ST25 is hereby incorporated by reference in its entirety.
Millions of North Americans suffer from irreversible vision loss due to retinal degenerative diseases such as retinitis pigmentosa, age-related macular degeneration, cone-rod dystrophies, Leber congenital amaurosis, Stargardt disease, and Usher syndrome. The common cause of sight impairments in these diseases is the progressive death of the light-sensing cells of the retina; the rod and cone photoreceptors. While rod photoreceptor degeneration leads to night blindness and reduced peripheral vision, it is the loss of cones that is the most devastating to patients as these cells provide the most-important daylight and high acuity macular vision in humans. Notably, even in diseases that affect rods due to mutations in rod genes (e.g., retinitis pigmentosa), the degeneration of rods eventually leads to the loss of cones at late stages of the disease. Considering the importance of cone photoreceptors for daylight vision, this secondary loss of cones is a major clinical problem. Although there are currently some treatments available to slow disease progression and cone loss in certain conditions (e.g., anti-VEGF therapy for wet macular degeneration), there are no cures available to restore normal vision for any retinal degenerative diseases. Since the incidence of age-related retinal degeneration is expected to increase drastically in coming years due to the aging population, new therapies are urgently needed.
One possibility to restore vision would be to replenish the lost photoreceptor cells. The preferred approach to achieve this has been with photoreceptor transplantation, for which considerable advances have been made in the last 10 to 15 years (reviewed by Santos-Ferreira et al., 2017). However, there has been a recent set back in the field with the important finding that the vast majority of what was originally thought to be “integrated” photoreceptors were actually host cells that had taken up the fluorescent reporter from the transplanted cells (Ortin-Martinez et al., 2016; Pearson et al., 2016; Santos-Ferreira et al., 2016; Singh et al., 2016). These studies revealed that the integration efficiency of transplanted cells was much lower than previously interpreted, raising concerns on whether transplantation approaches are even possible in the retina. New avenues of research are consequently required to bypass this integration limitation for photoreceptor regeneration.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present disclosure exploits an endogenous source of cells to regenerate photoreceptors for use within the retina. The present disclosure reports the generation (production) of neurons (e.g., cone photoreceptors-like cells) ex vivo by modifying mammalian neuroepithelial cells so that they recombinantly express IKAROS Family Zinc Finger 4 (Ikzf4). It also reports the generation (production) of neurons (e.g., cone photoreceptors-like cells) in vitro, ex vivo, and in vivo by modifying mammalian glial cells (e.g., Müller glia cells) so that they recombinantly co-express IKAROS Family Zinc Finger 1 (Ikzf1) and IKAROS Family Zinc Finger 4 (Ikzf4).
More specifically, in accordance with the present disclosure, there are provided the following items:
Item 1. A recombinant nervous system cell comprising nucleic acid encoding IKAROS Family Zinc Finger 4 (Ikzf4) and/or IKAROS Family Zinc Finger 1 (Ikzf1).
Item 2. The recombinant cell of item 1, which is a retinal cell.
Item 3. The recombinant cell of item 2, comprising nucleic acid encoding Ikzf4.
Item 4. The recombinant cell of any one of items 1-3, which is a neuroepithelial cell.
Item 5. The recombinant cell of any one of items 1-3, which is a glial cell.
Item 6. The recombinant cell of item 5, which is a Müller cell.
Item 7. The recombinant cell of any one of items 1-3, which is a neuron.
Item 8. The recombinant cell of any one of items 1-7, which expresses Ikzf4 and Ikzf1.
Item 9. The recombinant cell of item 8, which is a cone photoreceptor.
Item 10. The recombinant cell of any one of items 1-9, wherein the nucleic acid is operably linked to a glial specific promoter.
Item 11. The recombinant cell of any one of items 1-10, wherein the nucleic acid is comprised in an adeno-associated vector (AAV).
Item 12. The recombinant cell of item 11, wherein the AAV is of the Shh10 serotype.
Item 13. The recombinant cell of any one of items 1-10, wherein the nucleic acid is comprised in a lentiviral vector.
Item 14. A cell population comprising the cell defined in any one of items 1-13.
Item 15. A vector comprising a glial specific promoter operably-linked to a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) and/or a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4).
Item 16. The vector of item 15, comprising Ikzf1.
Item 17. The vector of item 15 or 16, comprising Ikzf4.
Item 18. The vector of any one of items 15-17, which is an adeno-associated viral vector (AAV).
Item 19. The vector of item 18, which is of the Shh10 serotype.
Item 20. The vector of any one of items 15-17, which is a lentiviral vector.
Item 21. A pharmaceutical composition comprising (a)(i) a nucleic acid encoding IKAROS Family Zinc Finger 1 (Ikzf1); and/or a nucleic acid encoding IKAROS Family Zinc Finger 4 (Ikzf4); or (ii) the vector defined in any one of items 14-19; and (b) a pharmaceutically acceptable carrier.
Item 22. A transgenic non-human animal comprising the recombinant nervous system cell defined in any one of items 1-13; or the vector defined in any one of items 15-20.
Item 23. A method of producing a recombinant cone photoreceptor, comprising:
(a) introducing a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) in a Müller glia cell; and
introducing a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4) in the Müller glia cell; or
introducing a nucleic acid molecule encoding Ikzf4 in a retinal neuroepithelial cell;
whereby the retinal neuroepithelial cell or the Müller glia is reprogrammed into a recombinant cone photoreceptor.
Item 24. The method of item 23, wherein the introducing of (a) and (b) or (B) is ex vivo.
Item 25. The method of item 23, wherein the introducing of (a) and (b) or (B) is in vivo in a mammalian subject in need thereof.
Item 26. The method of any one of items 23-25, wherein the introducing of (a) and (b) or (B) is intraocular.
Item 27. The method of any one of items 23-26, wherein each of the nucleic acid molecules of (a) and (b) is in a vector.
Item 28. The method of any one of items 23-27, wherein the introducing of (a) and (b) is performed by electroporation.
Item 29. The method of any one of items 23-27, wherein the introducing of (a) and (b) is performed by viral-based gene delivery.
Item 30. The method of item 29, wherein the viral-based gene delivery is an adeno-associated virus (MV) gene delivery.
Item 31. The method of item 30, wherein the AAV is of the ShH10 serotype.
In other embodiments, there is provided a use of (a) a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) for introduction in a Müller glia cell; and of a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4) for introduction in the Müller glia cell; or (b) a nucleic acid molecule encoding Ikzf4 for introduction in a retinal neuroepithelial cell, whereby the retinal neuroepithelial cell or the Müller glia is reprogrammed into a recombinant cone photoreceptor.
In other embodiments, there is provided (a) a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) and of a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4) for their use in reprogramming a Müller glia cell into a recombinant cone photoreceptor; or (b) a nucleic acid molecule encoding Ikzf4 for its use in reprogramming a retinal neuroepithelial cell into a recombinant cone photoreceptor.
In other embodiments, there is provided a use (a) of a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) and of a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4) for their use in reprogramming a Müller glia cell into a recombinant cone photoreceptor; or (b) of a nucleic acid molecule encoding Ikzf4 for its use in reprogramming a retinal neuroepithelial cell into a recombinant cone photoreceptor.
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
Definitions
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
Herein, the term “about” has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Cells
In a one aspect, the present disclosure relates to a recombinant nervous system cell (e.g., mammalian such as human) expressing Ikzf1 and/or Ikzf4. As used herein the terms “nervous system cell” refers to neuroepithelial cells, glial cells and neurons. In accordance with the present disclosure, recombinant nervous system cells (e.g., neuroepithelial cells, glial cells) that are manipulated (e.g., cells transformed or transfected) to express Ikzf1 and/or Ikzf4 will become (i.e., be reprogrammed as) neurons, as a result of this expression. For example, and without being so limited, recombinant neuroepithelial cells that are manipulated (e.g., cells transformed or transfected) to express Ikzf4 cells will become cone photoreceptor (see e.g., Examples 2-3) and Müller glia cells that are manipulated (e.g., cells transformed or transfected) to express Ikzf1 and Ikzf4 will become cone photoreceptor cells (see e.g., Examples 4-10).
In an embodiment, nervous system cells targeted by methods described herein are endogenous retinal nervous system cells of a subject in need for cone photoreceptors. In this embodiment, vectors of the present disclosure are introduced in the eye(s) of the subject in need thereof and the targeted cells are reprogrammed in vivo. Alternatively, in other embodiments, recombinant cells are reprogrammed ex vivo or in vitro. For such embodiments of the methods described herein, sources of nervous system cells can be embryonic nervous system cells (e.g., embryonic neuroepithelial cells), adult nervous system cells (e.g., adult Müller glia cells can be isolated from postmortem human tissue), embryonic stem cells transformed into nervous system cells such as neuroepithelial cells by the Zhong et al. 2014 method, or induced pluripotent stem cells (IPSCs) transformed into nervous system cells such as neuroepithelial cells by the Nakano et al. 2012 method.
In a specific embodiment, the recombinant nervous system cell is a retinal nervous system cell. As used herein the term “retinal nervous system cell” refers to retinal neuroepithelial cells, retinal glial cells and retinal neurons. In specific embodiments, such cells can be adult cells.
In another specific embodiment, the recombinant nervous system cell is a glial cell (e.g., Müller glia cell).
In another specific embodiment, the recombinant nervous system cell is a neuron (e.g., cone photoreceptor). In another specific embodiment, the recombinant nervous system cell is a cone photoreceptor. In another embodiment it is a cell having cone morphologies and expresses at least one of (at least two of, or at least three of, or all four of) cone arrestin, RxRγ, S-opsin and PNA.
The term “recombinant” in the expression “recombinant retinal neuron cell” refers to a cell that has been genetically modified (e.g., transformed or transfected) to express Ikzf1 and Ikzf4.
IKAROS Family Zinc Finger 1 (Ikzf1) and IKAROS Family Zinc Finger 4 (Ikzf4) are transcriptions factors that belong to the family of zinc-finger DNA-binding proteins associated with chromatin remodeling. Ikzf1 is known to open chromatin (Bottardi S, Mavoungou L, Pak H, et al. The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis. PLoS Genet. 2014; 10(12):e1004827. Published 2014 Dec. 4. doi:10.1371/journal.pgen.1004827). As shown herein Ikzf4 is able to induce cone production.
As used herein, the term “Ikzfr1” refers to a biologically active Ikzf1 and unless the context suggests otherwise, encompasses any functional isoform of the Ikzf1 including, without being so limited in e.g., those depicted in human Uniprot Q13422-1, Q13422-2, Q13422-3, Q13422-4, Q13422-5, Q13422-6, Q13422-7 and Q13422-8 or any orthologue thereof e.g., mouse) (see also e.g.,
As used herein, the term “Ikzf4” refers to a biologically active Ikzf4 and unless the context suggests otherwise, encompasses any functional isoform of the Ikzf4 including, without being so limited in e.g., those depicted in human Uniprot Q9H2S9-1 and Q9H2S9-2 or any orthologue thereof (e.g., mouse) (see e.g.,
The instant disclosure encompasses the use of Ikzf1 and Ikzf4 that can differ from the native proteins (e.g., human and other mammalian orthologues). For instance, proteins can be used that satisfy the consensus sequences derived from the alignments in
Other functional Ikzf1 and Ikzf4 variants may also be obtained by deletion of 1, 2, 3, 4, 5, 10, 15 or 10 and up to 30, 40, 50 or 60 amino acids of the native or sequences satisfying the consensus Ikzf1 and Ikzf4 sequences e.g., at the N-terminal end and/or the C-terminal end of these protein, preferably the N-terminal end. Similarly, protein construct comprising Ikzf1 and Ikzf4 may also encompass additional amino acids (1, 2, 3, 4, 5, 10, 15 or 10 and up to 30, 40, 50 or 60 amino acids) at the N- and/or C-terminal of the native or sequences satisfying the consensus Ikzf1 and Ikzf4 sequences. Such additional amino acids may be the result of cloning or could be added to increase the stability or targeting of the proteins.
Nucleic Acids, Vectors, Cells
The present disclosure also relates to nucleic acids comprising nucleotide sequences encoding the above-mentioned Ikzf1 and/or Ikzf4. The nucleic acid can be a DNA or an RNA. The nucleic acid sequence can be deduced by the skilled artisan on the basis of the disclosed amino acid sequences. In a specific embodiment, the nucleic acid is any one of the nucleic acid sequences depicted in
The Ikzf1 and/or Ikzf4 could also be modified for better expression/stability/yield in the cell; codon optimization for expression in the heterologous nervous system cell such as glial cells (e.g., Müller glia cell); use of different combinations of promoter/terminators for optimal co-expression of multiple nucleic acids.
A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. Conservative amino acid mutations may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).
In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may be a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term “basic amino acid” it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term “hydrophobic amino acid” (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (He or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). “Acidic amino acid” refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).
Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2, BLAST-P, BLAST-N, COBALT or FASTA-N, or any other appropriate software/tool that is known in the art (Johnson, et al. 2008).
The substantially identical sequences of the present disclosure may be at least 75% identical; in another example, the substantially identical sequences may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical at the amino acid level to sequences described herein.
In another aspect, the present disclosure relates to a vector comprising a promotor operably-linked to a nucleic acid molecule encoding Ikzf1 and/or a nucleic acid molecule encoding Ikzf4.
The vectors can be of any type suitable, e.g., for expression of said polypeptides or propagation of genes encoding said polypeptides in a particular organism. The organism may be of eukaryotic origin (e.g., human).
The specific choice of vector depends on the host organism and is known to a person skilled in the art. In an embodiment, the vector comprises transcriptional regulatory sequences or a promoter operably-linked to a nucleic acid comprising a sequence encoding an Ikzf1 and/or Ikzf4 of the disclosure. A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. “Transcriptional regulatory sequences” or “transcriptional regulatory elements” are generic terms that refer to DNA sequences, such as initiation and termination signals (terminators), enhancers, and promoters, splicing signals, polyadenylation signals, etc., which induce or control transcription of protein coding sequences with which they are operably-linked.
Without being so limited, vectors useful to express the Ikzf1 and Ikzf4 of the present disclosure include any vector containing a glial (e.g., Müller cell)-specific promoter to drive expression of Ikzf1 and/or Ikzf4 or nonspecific promoters to drive expression of Ikzf1 and/or Ikzf4 in neuroepithelial cells; or, when certain viral vector serotypes are used, can target specifically Müller glia through the viral capsid. Many useful (human) cell expression vectors, are commercially available, e.g., from Addgene, Invitrogen (www.lifetechnologies.com), the American Type Culture Collection (ATCC; www.atcc.org) or the Euroscarf collection (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/).
Promoters useful to express the Ikzf1 and/or Ikzf4 of the present disclosure include glial-specific promoters Slc1a3 (solute carrier family 1 (glial high-affinity glutamate transporter, member 3), also called Glutamate Aspartate Transporter (GLAST)) promoter, Lhx2 promoter, and Sox9 promoter. Promoters useful to express the Ikzf1 and/or Ikzf4 of the present disclosure in cells such as neuroepithelial cells include nonspecific promoters such as CAG and CMV.
Without being so limited, in certain embodiments, it may be useful to include in the constructs disclosed herein means to reduce or stop expression of Ikzf1 and/or Ikzf4 include Tet-On (expression only in the presence of tetracyclin/doxycyxlin whereas Tet-off is always expressed except when tetracyclin/doxycyxlin is present).
The term “heterologous coding sequence” refers herein to a nucleic acid molecule that is not normally produced by the host cell in nature.
A recombinant expression vector (plasmid, viral vector) comprising a nucleic acid molecule(s) of the present disclosure may be introduced into a cell, e.g., a Müller cell or a neuroepithelial cell, capable of expressing the protein coding region from the defined recombinant expression vector. Accordingly, the present disclosure also relates to cells (host cells) comprising the nucleic acid and/or vector as described above. The terms “host cell” and “recombinant cell” are used interchangeably herein. Such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny(ies) may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. Vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), Sambrook and Russell (supra) and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known and may be used to deliver the vector DNA of the disclosure to a subject for gene therapy.
In specific embodiments, as indicated above, the cells expressing Ikzf1 and/or Ikzf4 are mammalian nervous system cells such as neuroepithelial cells, glial cells (e.g., retinal glial cells) or neurons.
In another aspect, the present disclosure relates to a method of producing a recombinant cone photoreceptor, comprising: (a) introducing a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) in a Müller glia cell; and (b) introducing a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4) in the Müller glia cell, whereby the Müller glia is transformed into a recombinant cone photoreceptor. In specific embodiments, (a) and (b) can be in vitro, ex vivo or in vivo. The introduction/administration of (a) and (b) can be simultaneous or sequential in any order (i.e. (a) before (b) or (b) before (a). When administration is simultaneous, a single nucleic acid (vector) can be used to encode both Ikzf1 and Ikzf4. When the introducing (a) and (b) is in vivo, the subject may be a subject in need thereof.
As used herein the terms “sequential” in the context of introducing or administering (a) and (b) sequentially refers to successive introduction or administration of (a) and (b). In specific embodiments, the two introductions or administration may be separated by about 1 week.
In another aspect, there is provided a method of preventing or treating a disease or condition associated with a cone photoreceptor degeneration or a symptom thereof, comprising: (a) administering a nucleic acid molecule encoding IKAROS Family Zinc Finger 1 (Ikzf1) in a Müller glia cell; and (b) administering a nucleic acid molecule encoding IKAROS Family Zinc Finger 4 (Ikzf4) in the Müller glia cell, to a subject in need thereof. The nucleic acids are advantageously administered in a therapeutically effective amount.
As used herein the term “disease or condition associated with cone photoreceptor degeneration” refers to retinal degenerative diseases such as retinitis pigmentosa, age-related macular degeneration, cone-rod dystrophies, Leber congenital amaurosis, Stargardt disease, and Usher syndrome. As used herein the term “or a symptom thereof” refers as least to the degeneration of cone photoreceptor including a reduction in cone photoreceptor number and/or activity or a reduction in vision.
The introduction or administering of (a) and/or (b) (route of administration) can be intraocular such as but not limited to intravitreal or sub-retinal.
As used herein the term “subject” is meant to refer to any mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In a particular embodiment, it refers to a human.
As used herein, the term “subject in need thereof” in the above-disclosed methods is meant to refer to a subject that would benefit from receiving a nucleic acid molecule encoding Ikzf1 and a nucleic acid molecule encoding Ikzf4 in a Müller glia cell in accordance with the present disclosure (e.g., for introduction into Müller glia cell by e.g., intravitreal or sub-retinal administration). In specific embodiments, it refers to a subject that already has a disease or condition associated with a cone photoreceptor degeneration or a symptom thereof. In another embodiment it further refers to a subject that has as retinitis pigmentosa, age-related macular degeneration, cone-rod dystrophies, Leber congenital amaurosis, Stargardt disease, and Usher syndrome.
As used herein, the term “prevent/preventing/prevention” or “treat/treating/treatment”, refers to eliciting the desired biological response, i.e., a prophylactic and therapeutic effect, respectively in a subject. In accordance with the present disclosure, the therapeutic effect comprises one or more of a decrease/reduction in the severity, intensity and/or duration of the disease or condition associated with a cone photoreceptor degeneration or a symptom thereof (referred to hereinafter in the present paragraph as “disease, condition or any symptom thereof”) following administration of the nucleic acids, vectors (e.g., AAV), cells or pharmaceutical composition (“agent”) of the present disclosure when compared to its severity, intensity and/or duration in the subject prior to treatment or as compared to that/those in a non-treated control subject having the disease, condition or any symptom thereof. In accordance with the disclosure, a prophylactic effect may comprise a delay in the onset of the disease, condition or any symptom thereof in an asymptomatic subject at risk of experiencing the disease, condition or any symptom thereof at a future time; or a decrease/reduction in the severity, intensity and/or duration of disease, condition or any symptom thereof occurring following administration of the agent of the present disclosure, when compared to the timing of their onset or their severity, intensity and/or duration in a non-treated control subject (i.e. asymptomatic subject at risk of experiencing the disease, condition or any symptom thereof); and/or a decrease/reduction in the progression of any pre-existing disease, condition or any symptom thereof in a subject following administration of the agent of the present disclosure when compared to the progression of the disease, condition or any symptom thereof in a non-treated control subject having such pre-existing disease, condition or any symptom thereof. As used herein, in a therapeutic treatment, the agent of the present disclosure is administered after the onset of the disease, condition or any symptom thereof. As used herein, in a prophylactic treatment, the agent of the present disclosure is administered before the onset of the disease, condition or any symptom thereof or after the onset thereof but before the progression thereof.
Combination
In addition to nucleotide sequences encoding the above-mentioned Ikzf1 and/or Ikzf4, other factors can be used in accordance with the methods disclosed herein could enhance differentiation of the reprogrammed cells into mature cone photoreceptors, including, without being so limited, factors involved in cone differentiation, survival, chromatin remodelling, and proliferation, either in the form of co-administered or sequentially administered nucleic acids encoding such factors or as co-administered or sequentially administered small molecules, proteins, etc. In specific embodiments, the recombinant cell disclosed herein comprise heterologous nucleic acid encoding Ikzf1 and/or Ikzf4, and one more heterologous nucleic acid encoding one of the above factors, or 2 or less of these factors, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less additional heterologous nucleic acid heterologous nucleic acid encoding one of the above factors. As used herein, the term “heterologous” refers to nucleic acid that was voluntarily introduced in the host cell (endogenously or exogenously) as disclosed herein.
Dosage
Any amount of the nucleic acids, vectors, cells or pharmaceutical compositions disclosed herein (“agent”) can be administered to a subject. The dosages will depend on many factors including the mode of administration and the age of the subject. Typically, the amount of agent of the disclosure contained within a single dose will be an amount that effectively prevent, or treat a disease or condition associated with a cone photoreceptor degeneration or a symptom thereof without inducing significant toxicity. As used herein the term “therapeutically effective amount” is meant to refer to an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. The dose varies with the type of administration, Typically, the agent in accordance with the present disclosure can be administered to subjects in doses ranging from 0.001 to 500 mg (of nucleic acid, viral particle or composition comprising either with a pharmaceutically acceptable carrier)/per eye and, in a more specific embodiment, about 0.1 to about 100 mg/per eye, and, in a more specific embodiment, about 0.2 to about 20 mg/per eye, and in a more specific embodiment, about 0.2 to about 10 mg/per eye.
In mice for example, when electroporation was used, 1 μl of DNA solution was administered at 3 μg/μl/eye (i.e. 3 μg (0.003 mg) of DNA/eye). When viral-gene therapy was used (i.e. AAV), 2 μl/eye of ShH10-Ikzf1 at a titer of 6,96E+12 vg/ml and 2 μl/eye of ShH10 Ikzf4 at a titer of 5,87E+13 vg/ml. The allometric scaling method of Mahmood et al. (Mahmood et al. 2003) can be used to extrapolate the dose from mice to human. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient.
The therapeutically effective amount of the agent of the instant disclosure may also be measured directly. Typically, a pharmaceutical composition of the disclosure can be administered in an amount from about 0.001 mg up to about 500 mg per eye as a single dose (e.g., 0.05, 0.01, 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 50 mg, 100 mg, or 250 mg). In specific embodiment, the action of the dose is applied for about one month.
These are simply guidelines since the actual dose must be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient or by a nutritionist. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient as indicated above and other clinically relevant factors. In addition, patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that an agent in accordance with the instant disclosure is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
Carriers/Vehicles
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, physiological media, and the like that are physiologically compatible. In embodiments, the carrier is suitable for ocular administration. Pharmaceutically acceptable carriers for ocular administration include sterile aqueous solutions (e.g., saline) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents, such as for ocular application, is well known in the art. Except insofar as any conventional media or agent is incompatible with the compounds of the disclosure, use thereof in the compositions of the disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Administration and Introduction
The above-mentioned nucleic acids or vectors may be delivered to cells in vivo (to induce the expression of the Ikzf1 and Ikzf4 in accordance with the present disclosure) using methods well known in the art such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid-based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo. A delivery apparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor. Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes. In specific embodiment, the vector(s) comprise a system to turn off Ikzf1 and/or Ikzf4 after a specific time period after administration (e.g., tetracycline-inducible promoters, which are turned off once tetracycline is removed).
As used herein, the term “decrease” or “reduction” (e.g., of a disease or condition associated with a cone photoreceptor degeneration or of a symptom thereof) refers to a reduction of at least 10% as compared to a control subject (a subject not treated with an agent of the present disclosure), in an embodiment of at least 20% lower, in a further embodiment of at least 30% lower, in a further embodiment of at least 40% lower, in a further embodiment of at least 50% lower, in a further embodiment of at least 60% lower, in a further embodiment of at least 70% lower, in a further embodiment of at least 80% lower, in a further embodiment of at least 90% lower, in a further embodiment of 100% (complete inhibition).
Similarly, as used herein, the term “increase” or “increasing” (e.g., of an Ikzf1 and/or Ikzf4 biological activity in a method of the present disclosure of at least 10% as compared to a control, in an embodiment of at least 20% higher, in a further embodiment of at least 30% higher, in a further embodiment of at least 40% higher, in a further embodiment of at least 50% higher, in a further embodiment of at least 60% higher, in a further embodiment of at least 70% higher, in a further embodiment of at least 80% higher, in a further embodiment of at least 90% higher, in a further embodiment of 100% higher, in a further embodiment of 200% higher, etc. The “control” for use as reference in the method disclosed herein of preventing or treating a disease or condition associated with a cone photoreceptor degeneration or of a symptom thereof may be e.g., a control subject that has a disease or condition associated with a cone photoreceptor degeneration or of a symptom thereof, and that is not treated with an agent present disclosure.
The nucleic acids disclosed herein could be advantageously delivered through gene therapy.
A “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts and may be used for gene therapy as well as for simple protein expression. “Gene delivery,” “gene transfer,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
A “viral vector” is defined as a recombinantly produced virus or viral; particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adeno-associated virus vectors (see e.g., Example 10 and
In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. AAVs include more than 10 serotypes. In a specific embodiment, the MV serotype Shh10 which harbors a Müller-cell specific capsid is used (see e.g.,
Recombinant cone photoreceptors as disclosed herein could be used in therapy for transplantation in the eyes of subjects in need thereof or be used as a research tool for drugs and other treatments and transfection conditions.
The present disclosure is illustrated in further details by the following non-limiting examples.
Animals. Animal work was performed in accordance with the Canadian Council on animal care and IRCM guidelines. GlastCreERT mice (stock 012586) and the RosaYFPfl/fl reporter mice (stock 006148) were obtained from The Jackson Laboratory. GlastCreERT mice is a BAC transgenic line expressing CreERT under the control of the Slc1a3 (solute carrier family 1 (glial high-affinity glutamate transporter, member 3), also called Glutamate Aspartate Transporter (GLAST)) promoter. When crossed with a strain containing a loxP site flanked sequence, the offspring are useful for generating tamoxifen-induced, Cre-mediated recombination of DNA regions specifically in glial cells in the adult or progenitor cells in the embryo. The RosaYFPfl/fl mutant mice have a loxP-flanked STOP sequence followed by the Yellow Fluorescent Protein gene (YFP) inserted into the Gt(ROSA)26Sor locus. When bred to mice expressing Cre recombinase, the STOP sequence is deleted and EYFP expression is observed in the cre-expressing tissue(s) of the double mutant offspring. These mutant mice may be useful in monitoring the activity of Cre in living tissues and tracing the lineage of cells that have expressed Cre in embryos, young, and adult mice at desired time points.
DNA constructs. PCALL2, a conditional targeting vector, was obtained from Pierre Mattar (and originally from Dr. Corrine Lobe https://health.uconn.edu/mouse-genome-modification/resources/conditional-knock-outexpression-vectors) and digested with Clal and Sphl to insert mCherry, a fluorophore, (amplified from MSCV-mCherry) in the Loxp cassette. IRES-EGFP was removed with Smal and Notl digestions. A Gateway cassette was added within the multiple cloning site (MCS) for some gene sequence insertions with Gateway Cloning System (Thermo Fisher), while others were inserted directly in the MCS by restriction digestions or with In-Fusion cloning (Clontech). Ikzf1 was obtained from Dr. Georgopoulos. Caz1v2 and Pou2f1 sequences were generated by Dr. Mattar and Ikzf4 by Christine Jolicoeur. Pou2f2 was obtained from IMAGE™ (40046279). Brn2, Ascl1, and Myt1l sequences were amplified from plasmids obtained from Addgene (#27151, 27150, and 27152 respectively). Apobec2b was provided by Dr. Di Noia.
Ex vivo work. Eyes from post-natal days 0-1 (P0-1). GlastCreERT;RosaYFPfl/fl mice were collected in PBS under sterile conditions. Vectors (3 ug/ul) described above were injected sub-retinally and a current (50 millisec duration, 950 millisec interval, 40-50 volts, unipolar electrodes; BTX ECM 830) was applied over the eye with the positive electrode facing the cornea. Retinas were then dissected out in PBS and placed on a culture insert (Millicell) in a 6-well plate (Flacon) containing 1.3 ml of equilibrated media (DMEM with 10% FBS and 1× pen/strep; Gibco). Explants were left in 5% CO2 incubator with 90% humidity for the duration of the culture, with media-change 3 times per week. At DIV12 (Days in vitro 12), hydroxytamoxifen (Cayman Chemical Co., cat #13258-1) was added to culture media at a final concentration of 5 uM and EGF (PreproTech) at a concentration of 100 ng/mL and were kept in media until DIV14/15. When indicated, 2′-Deoxy-5-ethynyluridine (EdU) (Abcam), a DNA synthesis monitoring probe, was added to the media at a concentration of 10 ug/ml at DIV12, 15, 18, and/or 21 and left for 3 days. At DIV26, media was removed from the well and replaced with 1 ml of 4% Paraformaldehyde (PFA; Electron microscopy sciences) for 5 minutes at room temperature. 1 ml 4% PFA was then added over the culture insert and left for another 5-minute incubation at room temperature. Explants were quickly washed with PBS and left in 20% sucrose in PBS at 4° C. for 2-5 hours before being removed from the culture insert with curved forceps and frozen in a 20% sucrose:OCT (Sakura) solution for cryosectionning.
In vivo work. Wild-type or GlastCreERT;RosaYFPfl/fl P0-2 mice were anesthetized on ice, injected sub-retinally with 1 ul of DNA vectors (3 ug/ul) in 1 eye and subjected to an electrical current (50millisec duration, 950 millisec interval, 80 volts, unipolar electrodes) over the eyes with the positive electrode over the injected eye. When indicated, some animals were injected intraperitoneally with EdU (Abcam) from P3-7 to label cells that have undergone S-phase during this period. From P21-23 inclusively, the animals were injected intraperitoneally daily with 90 ug of tamoxifen (Toronto Research Chemicals and Cedarlane Labs) per gram of body weight. Animals were euthanized by CO2 between P37-P56. Eyes were collected, fixed for 5 min in 4% PFA at room temperature, washed with PBS, and left in 20% sucrose for 4-6 hours at 4° C. before being frozen in 20% sucrose:OCT for cryosectionning.
Immunohistochemistry. Blocks were cryostat (Leica)-sectioned at 25 μm. Slides were incubated in PBS for 2 minutes to remove OCT and left in blocking solution (PBS, 3% BSA (Sigma), and 0.3% triton-100×(Sigma)) for 1 hour at room temperature. They were then incubated in primary antibody solution (in blocking) overnight at room temperature (see Table.1 below for antibody list).
This was followed with 3 washes in PBS and secondary antibody incubation in PBS for 1 hour at room temperature. The slides were washed again with PBS and incubated with Hoechst ( 1/10,000 in PBS; Molecular probes) for 5 minutes at room temperature. The slides were then washed and mounted with Mowiol or underwent EdU click-it (Abcam) reaction following the company's protocol (modified to use ½ of recommended B-component in order to reduce potential bleed-through of AlexaFluor-647).
Lentivirus production To produce lentivirus, 293FT cells (Thermo Fishes Scientific) were plated onto 10 cm dishes (Corning). When plates were 70% confluent, transfection media was produced. Transfection media consisted of 1 ml of DMEM (Gibco) with 5 ug of psPAX2 (Addgene, Cat.Nr. 12260), 10 ug of pMD.2G (Addgene, Cat.Nr. 12259), 10 ug of plasmid of interest and 45 ul of PEI (Polyethylenimine, Polysciences). After adding PEI, the transfection media was left to incubate for 15 minutes at room temperature and then was added dropwise to the cell dish. 6 hours after adding transfection media, cell media was replaced with fresh DMEM supplied with 5% BSA (Sigma-Aldrich). Lentiviral collection and spindown was performed at 24 h and 48 h after initial media change by using Lenti-X-concentrator (Clontech) with the according protocol (Clontech, PT4421-2). Lentiviral titer was determined by using the Lenti-X qRT-PCR Titration Kit (Clontech).
Müller glia culture. Müller glia were cultured from P8-10 CD1 wild-type mice following a previously published protocol (Liu et al., 2017) and were passaged 3 times before being seeded in 24-well plates containing coverslips coated with 0.1% bovine gelatin (Sigma-Aldrich). 24 h after seeding, media was replaced with 500 ul per well of lentiviral media (containing LV-M2-rtTA; LV-tet-Ikzf1; LV-tet-Eos at each MOI 10) supplied with 8 ug/ml of Polybrene (Sigma-Aldrich) and spinfected for 1 h at 2000 rpm. 1-day post-infection (dpi), lentiviral media was exchanged with full DMEM supplemented with 2 ug/ml of doxycycline (dox, Sigma-Aldrich). Half of the media was exchanged with new dox-supplemented full DMEM every 2-3 days. At 9 dpi, until 21 dpi, half of the media was switched every 2-3 days with retinal maturation medium (Gonzalez-Cordero et al., 2017) supplemented with 2 ug/ml dox. At 21 dpi, cells were fixed in 4% PFA (Electron Microscopy Sciences) for 15 min at room temperature or lysed in RLT buffer (Qiagen) for RNA isolation and qPCR.
RNA isolation and Quantitative PCR. Retinal explants were dissociated with 100 units of papain (Worthington, LS003124). GFP+ cells were FAC-sorted from the dissociated retinal explants 6 days after electroporation. Collected cells were sorted directly into Qiagen™ Buffer RLT plus and RNeasy™ microkit (Qiagen, 74004) was used to isolate RNA from the cells as instructed by the manufacturers protocol. Isolated RNA was reverse transcribed using Superscript™ VILO Master Mix (Thermofisher Scientific, 11755050). cDNA was amplified by quantitative PCR using SYBR™ Green Master mix (Thermofisher Scientific, A25742). Primers used were NrI pF: CGAGCAGTGCACATCTCAGTTC (SEQ ID NO: 69), pR: AACTGGAGGGCTGGGTTACC (SEQ ID NO: 70), Nr2e3 pF: AAGCTCCTGTGTGACATGTTCAA (SEQ ID NO: 71), pR: AAGCTCCTGTGTGACATGTTCAA (SEQ ID NO: 72).
Adeno associated viruses. Viral vectors (see
Microscopy and cell counts. All images were obtained by SP8 confocal microscopy (Leica), analyzed on Volocity™ software (Perkin Elmer), and processed on Fiji™ (ImageJ), and Adobe™ Illustrator (Adobe). For explant cell count, YFP+ mCherry+ cells were analyzed unless specified that only Ikzf1/4 morphologically reprogrammed cells were analyzed, which corresponds to YFP+ mCherry+ cells with round or cone-like morphologies.
Statistics. Statistical analyses were performed with Prism (GraphPad) software.
The expression of Ikzf4 was studied in the mouse retina during the temporal window of cone genesis. As the antibody specific to Ikzf4 was raised in the same species as the early cone marker antibody, the inventors could not investigate whether Ikzf4 co-localizes with Rxry, a marker for cone photoreceptors. To overcome this issue, Otx2, a marker for photoreceptor precursors at E15, was used. Since cone photoreceptors are born during the embryonic stages of mouse retinogenesis (Rapaport et al., 2004; Young, 1985a, b), the majority of the Otx2+ cells at this age are cone photoreceptor precursors. Expression of Ikzf4 was detected in the retinal progenitor layer, and in some Otx2+ cells (
The functional role of Ikzf4 in the developing retina was next investigated. It was tested whether Ikzf4 was sufficient to induce cone production in late-stage retinas, a stage at which no cones are normally generated. P0 retinal explants (i.e. neuroepithelial cells, namely multipotent cells) were electroporated with vectors expressing either only GFP (see
Next was assessed whether Ikzf4 overexpression leads to a reduction of mRNA levels of NrI and Nr2e3, two critical rod differentiation genes, the repression of which is known to lead to the generation of a retina composed of cone-like cells only (Mears et al, 2001). To test this, P0 retinas were electroporated with control GFP or Ikzf4-IRES-GFP and the GFP+ population were sorted after 6 days, mRNA isolated and RT-qPCR performed using primers specific to NrI and Nr2e3. A significant reduction of mRNA expression of both NrI and Nr2e3 was detected (
Taken together, these results suggest that Ikzf4 is sufficient to induce a repression of rod genes (i.e., NrI and Nr2e3) and induce cone production in late stage retinas.
Co-Expression of Ikzf1 and Ikzf4 can Reprogram Müller Glia Into Immature Cone-Like Cells Ex Vivo in Retinal Explants in Terms of Shape
The Müller-specific Cre mouse line Glast-CreERT, which also carried the RosaYFPfl/fl reporter (GlastCreERT;RosaYFPfl/fl), was used, allowing to lineage-trace all Müller-derived cells by imaging the YFP fluorescence. Retinas were electroporated at postnatal day 0-1 (P0/1) with Cre-dependent expression constructs containing mCherry, a fluorophore, ((pCAG-loxP-mCherry-stop-loxP-gene (
It was noticed that mCherry continued to be expressed within Müller glia that had activated Cre, allowing to focus the analysis on electroporated Müller cells (YFP co-labelling with mCherry). The genes screened were Ikzf1 (
Out of 23 gene expression combinations screened (see
In addition to morphology changes, the majority (61.1%) of YFP+ mCherry+ cells in the Ikzf1/4 condition moved to the apical side of the retina (in the ONL), where cone photoreceptors are usually located (
Furthermore, within the Ikzf1/4 expressing population, the observed change in morphology was associated with a re-localization to the apical side of the ONL: whereas only 3% of Müller-like cells located to the apical side of the ONL, 91.3% of round cells, and 79.9% of cone-like cells were found there (
To analyze whether these morphologically reprogrammed cells (cone-like and round population) kept their Müller identity, immunofluorescence were performed for the Müller glia markers Lhx2, and Sox2 (
It was next assessed whether the reprogrammed cells expressed photoreceptor markers by immunofluorescence (
It is important to note that single overexpression of either Ikzf1 or Ikzf4 did not induce this reprogramming. Indeed, Ikzf1 did not produce changes in Müller glia (Data not shown), at least to the extent analyzed, while Ikzf4 induced RxRγ expression, but did not change their morphology and very rarely induced downregulation of Müller glia markers (
To determine whether Ikzf1/4-expressing Müller glia proliferate before reprogramming to cone-like cells ex vivo, EdU time course experiments (EdU being the proliferation marker) were performed spanning DIV12-24, which corresponds to the time point at which is added hydroxytamoxifen, all the way to 2 days before fixation.
One set of experiments spanned DIV12-15 and DIV15-18 (
It was next tested whether Ikzf1 and Ikzf4 expression would be sufficient to reprogram Müller glia in culture assays. Müller cell cultures were prepared following a published protocol (Liu et al., 2017) and infected with Ikzf1- and Ikzf4-expressing lentiviral vectors. The cells were cultured in a medium supplemented with taurine and retinoic acid, which were previously reported to promote photoreceptor maturation (Altshuler et al., 1993; Kelley et al., 1994).
Four weeks later, some RxRγ+ s-opsin+ cells were observed by immunofluorescence and gene induction was detected by RT-qPCR (
In order to test whether Ikzf1/4 expression could also reprogram Müller glia in vivo, the Cre-dependent Ikzf1/4 (pCAG-loxP-mCherry-Stop-loxP-Ikzf1/4; Pcall, same vectors as used in ex vivo experiments above; See
CreERT was activated with 3 consecutive tamoxifen injections from P21-P23, permanently labelling Müller glia and any derived progeny with YFP and initiating the expression of Ikzf1/4 in these cells (
To investigate whether the reprogrammed cells could survive in the retina, the above in vivo experiment was repeated and animals were sacrificed 5 weeks post-tamoxifen (
As an additional lineage tracing method and to exclude the possibility of YFP transfer, the previous in vivo protocol was repeated with intraperitoneal injections of EdU from P3-P7 (
AAVs have been previously used safely in humans and even in the eye for gene therapy (Petit et al., 2016). The Shh10 AAV serotype is mostly specific to Müller glia when injected intravitreally in the retina (Pellissier et al., 2014), although infection of RGCs and sometimes photoreceptors depending on injection site was also observed.
The use of AAV for Müller glia reprogramming in vivo was tested (i.e. AAV-Ikzf1 (
It was first found that infecting adult retinas in vivo with AAV-Ikzf4 induced expression of Ikzf4 in a large proportion of Müller glia (
Müller glia reprogramming with these infections are currently tested for the production of cone-like cells. GlastCreERT;RosaYFP mice, previously injected with tamoxifen to active permanent YFP expression in Müller cells, are intravitreally injected with AAV-Ikzf1 and AAV-Ikzf4 1 week later or AAV-Tomato as control. They are then sacrificed 5-7 weeks later and analyzed for YFP+(Müller-derived) cones by immunofluorescence.
To test the function of the reprogrammed cones, membrane potential is recorded in response to light and the reactivity of the cone is compared to that of endogenous cones. Alpha ganglion cells within the electroporated regions are also analyzed to determine whether de novo cones connect with synaptic partners and integrate retinal circuitry. Müller glia are also reprogrammed in 2 mouse models of retinitis pigmentosa to test whether Müller-derived cones restore vision. Experiments described in Example 9 are repeated in GlastCreERT;RosaYFP;Pde6bRD1 mice. These mice were obtained from Jackson Laboratory (strain 000659) and have the RD1 mutation in Pde6b gene, which leads to rod photoreceptor cell death and blindness by P21. Cone photoreceptors also degenerate with barely any present by P100.
Another retinal degeneration model used is the intraperitoneal injection of the drug N-methyl-N-nitrosourea (MNU), which kills photoreceptors by 7 days after injection (Tao et al., 2015) Experiments described in Example 9 are repeated with an intraperitoneal injection of MNU 1 week before tamoxifen administration to effectively kill photoreceptor cells before reprogramming Müller glia in cones. Vision can then be tested with behavioral tests (e.g., visual water tests, optomotor reflex) and by electroretinogram recordings.
To obtain insights into the underlying mechanism of reprogramming, RNA and ATAC-sequencing of Ikzf1/4-expressing Müller cultures at different time points are performed, allowing to identify both the transcriptomic changes and chromatin remodelling (respectively) occurring during reprogramming. Of particular interest is whether Müller glia go through an intermediate progenitor state or directly transdifferentiate into cones. scRNA-sequencing of in vivo Ikzf1/4 reprogrammed cells is also underway to better characterise the Müller-derived cells. These experiments will also identify targets to enhance reprogramming efficiency, as well as survival, and maturation of the cone-like cells.
Enhancing Maturation of Cone-Like Cells
Transitory transfection methods are additionally tested to limit potential toxicity from continuous Ikzf1/4 overexpression to determine whether this will improve cell survival. These methods include the doxycycline-inducible Tet-On system, which drives expression of Ikzf1 and Ikzf4 only in the presence of doxycycline, allowing to turn on and off their expression, as well as Ikzf1 and Ikzf4 protein transfections which are degraded by the cells and thus transiently present.
The scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.
This application is a PCT application Serial No CA2019/* filed on Nov. 5, 2019 and published in English under PCT Article 21(2), which itself claims benefit of U.S. provisional application Ser. No. 62/755,657, filed on Nov. 5, 2018. All documents above are incorporated herein in their entirety by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2019/051568 | 11/5/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62755657 | Nov 2018 | US |
