Patent Application
20180140720
The present disclosure illustrates that regeneration enhancer elements exist and can be isolated and provides guidance to new regeneration genes and tx regulators which may be relevant to the evolution of regenerative capacity. Further disclosed is that distinct tissues can engage different regeneration enhancers for the same gene and regeneration enhancers are minimal delivery modules for blocking/boosting regeneration with developmental factors. Finally, new tools are provided as well as enhancer delivery of regenerative factors.
Humans have a severely limited capacity to regenerate tissues like brain, heart, and limbs. Regenerative medicine aims to restore healthy tissue to structures like these, recapturing normal organ function. Proposed approaches to regenerative medicine include systemic administration of drugs, gene therapy using viruses or lipid vehicles, and cell therapy by systemic or local provision of stem cells. However, these approaches can suffer from low efficacy or potentially severe side effects. For example, the factor Neuregulin 1 can stimulate the division of cardiomyocytes, but also has the potential for neurological and oncogenic effects when delivered systemically. In addition, most gene regulatory elements used in gene therapy are constitutively active in all or specific cell types, but are not controlled in any way by the extent of injury. Thus, there are factors of safety and potential cytotoxicity through unwanted expression of genes in healthy tissues or continuing after the regeneration process is complete. Regulatory elements that activate only in injured tissue, and that ideally persistently activate expression until tissue is healed or regenerated, would provide a new level of specificity that could enhance gene therapy approaches to regenerative medicine.
Regenerative capacity is variable among species, tissues, and developmental stages. Whereas adult mammals do not regenerate cardiac muscle lost by ischemic myocardial infarction or amputated limbs, certain lower vertebrates like zebrafish regenerate these and other tissues like spinal cord very effectively. In a recent study, the inventors have established the concept of tissue regeneration enhancer elements, small DNA regulatory elements that help trigger regenerative programs in zebrafish. Importantly, it was also found that these elements can be engineered into simple DNA constructs that activate the production of pro-regenerative factors in injured and/or regenerative tissues in a manner that boosts regenerative capacity. These results support the idea that tissue regeneration enhance elements (TREEs) can be isolated from regenerating systems and engineered in a manner that can target therapeutic factors for regenerating tissue.
The present disclosure provides, in part, compositions and methods for tissue regeneration. One aspect of the present disclosure provides a gene therapy construct comprising, consisting of, or consisting essentially of a nucleic acid encoding one or more tissue regeneration enhancer elements (TREEs) operatively linked to a promoter. In some embodiments, the gene therapy construct further comprises a nucleic acid sequence comprising one or more pro- or anti-regenerative factors, the expression of which is under the influence of the tissue regeneration enhancer elements.
In other embodiments, the gene therapy particle comprises a vector system. In some embodiments, the vector system comprises an AAV vector system.
In some embodiments, the gene therapy construct further comprises a first and second AAV inverted terminal repeat (ITR) sequence flanking the sequence encoding the TREEs. In certain embodiments, the ITR nucleotide sequences are derived from AAV serotype 2 (AAV-2).
Another aspect of the present disclosure provides a pharmaceutical composition comprising the gene therapy construct provided herein in a biocompatible pharmaceutical carrier.
In another embodiment, the TREE comprises the lepb-linked regulatory enhancer element (LEN).
One aspect of the present disclosure provides a method of treating or ameliorating tissue repair comprising, consisting of, or consisting essentially of administering to a subject a therapeutically effective amount of gene therapy construct as provided herein such that the tissue repair is treated or ameliorated.
Another aspect of the present disclosure provides a method of augmenting tissue regeneration in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a gene therapy construct as described herein such that the tissue regeneration is augmented.
Another aspect of the present disclosure provides a method of delivering cell therapy to a subject comprising, consisting of, or consisting essentially of inserting within the genome of reprogrammed stem/progenitor cells of the subject a gene therapy construct as provided herein such that cell therapy is delivered.
Another aspect of the present disclosure provides a method for screening drugs that modulate regenerative capacity comprising, consisting of, or consisting essentially of transfecting a gene construct as described herein into a model system, administering to the system a drug of interest, and measuring regenerative capacity in response to the drug.
In some embodiments, the model system comprises a zebrafish model system.
Another aspect of the present disclosure provides all that is disclosed and illustrated herein.
The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying drawings, herein:
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
As used herein, the terms “gene transfer,” “gene delivery,” and “gene transduction” refer to methods or systems for reliably inserting a particular nucleotide sequence (e.g., DNA) into targeted cells.
As used herein, the term “gene therapy” refers to a method of treating a patient wherein polypeptides or nucleic acid sequences are transferred into cells of a patient such that activity and/or the expression of a particular molecule is restored.
As used herein, the term “adenoviral associated virus (AAV) vector,” “AAV gene therapy vector,” and “gene therapy vector” refer to a vector having functional or partly functional ITR sequences and transgenes. As used herein, the term “ITR” refers to inverted terminal repeats (ITR). The ITR sequences may be derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, and AAV-6. The ITRs, however, need not be the wild-type nucleotide sequences, and may be altered (e.g., by the insertion, deletion or substitution of nucleotides), so long as the sequences retain function to provide for functional rescue, replication and packaging. AAV vectors may have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes but retain functional flanking ITR sequences. Functional ITR sequences function to, for example, rescue, replicate and package the AAV virion or particle. Thus, an “AAV vector” is defined herein to include at least those sequences required for insertion of the transgene into a subject's cells. Optionally included are those sequences necessary in cis for replication and packaging (e.g., functional ITRs) of the virus.
The terms “adeno-associated virus inverted terminal repeats” or “AAV ITRs” refer to the palindromic regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. For use in some embodiments of the present invention, flanking AAV ITRs are positioned 5′ and 3′ of one or more selected heterologous nucleotide sequences. Optionally, the ITRs together with the rep coding region or the Rep expression products provide for the integration of the selected sequences into the genome of a target cell.
As used herein, the term “AAV rep coding region” refers to the region of the AAV genome that encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome. Muzyczka (Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-129 (1992)) and Kotin (Kotin, Hum. Gene Ther., 5:793-801 (1994)) provide additional descriptions of the AAV rep coding region, as well as the cap coding region described below. Suitable homologues of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al., Virol., 204:304-311 (1994)).
As used herein, the term “AAV cap coding region” refers to the region of the AAV genome that encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These cap expression products supply the packaging functions, which are collectively required for packaging the viral genome. In certain embodiments, AAV2 Cap proteins may be used.
As used herein, the term “AAV helper function” refers to AAV coding regions capable of being expressed in a host cell to complement AAV viral functions missing from the AAV vector. Typically, the AAV helper functions include the AAV rep coding region and the AAV cap coding region. The helper functions may be contained in a “helper plasmid” or “helper construct.” An AAV helper construct as used herein, refers to a molecule that provides all or part of the elements necessary for AAV replication and packaging. Such AAV helper constructs may be a plasmid, virus or genes integrated into cell lines or into the cells of a subject. It may be provided as DNA, RNA, or protein. The elements do not have to be arranged co-linearly (i.e., in the same molecule). For example, rep78 and rep68 may be on different molecules. An “AAV helper construct” may be, for example, a vector containing AAV coding regions required to complement AAV viral functions missing from the AAV vector (e.g., the AAV rep coding region and the AAV cap coding region).
As used herein, the terms “accessory functions” and “accessory factors” refer to functions and factors that are required by AAV for replication, but are not provided by the AAV vector or AAV helper construct. Thus, these accessory functions and factors must be provided by the host cell, a virus (e.g., adenovirus or herpes simplex virus), or another expression vector that is co-expressed in the same cell. Generally, the E1, E2A, E4 and VA coding regions of adenovirus are used to supply the necessary accessory function required for AAV replication and packaging (Matsushita et al., Gene Therapy 5:938 (1998)).
Portions of the AAV genome have the capability of integrating into the DNA of cells to which it is introduced. As used herein, “integrate,” refers to portions of the genetic construct that become covalently bound to the genome of the cell to which it is administered, for example through the mechanism of action mediated by the AAV Rep protein and the AAV ITRs. For example, the AAV virus has been shown to integrate at 19q13.3-qter in the human genome. The minimal elements for AAV integration are the inverted terminal repeat (ITR) sequences and a functional Rep 78/68 protein. The present invention incorporates the ITR sequences into a vector for integration to facilitate the integration of the transgene into the host cell genome for sustained transgene expression. The genetic transcript may also integrate into other chromosomes if the chromosomes contain the AAV integration site.
The predictability of insertion site reduces the danger of random insertional events into the cellular genome that may activate or inactivate host genes or interrupt coding sequences, consequences that limit the use of vectors whose integration is random, e.g., retroviruses. The Rep protein mediates the integration of the genetic construct containing the AAV ITRs and the transgene. The use of AAV is advantageous for its predictable integration site and because it has not been associated with human or non-human primate diseases, thus obviating many of the concerns that have been raised with virus-derived gene therapy vectors.
“Portion of the genetic construct integrates into a chromosome” refers to the portion of the genetic construct that will become covalently bound to the genome of the cell upon introduction of the genetic construct into the cell via administration of the gene therapy particle. The integration is mediated by the AAV ITRs flanking the transgene and the AAV Rep protein. Portions of the genetic construct that may be integrated into the genome include the transgene and the AAV ITRs.
The “transgene” may contain a transgenic sequence or a native or wild-type DNA sequence. The transgene may become part of the genome of the primate subject. A transgenic sequence can be partly or entirely species-heterologous, i.e., the transgenic sequence, or a portion thereof, can be from a species which is different from the cell into which it is introduced.
As used herein, the term “stably maintained” refers to characteristics of transgenic non-human primates that maintain at least one of their transgenic elements (i.e., the element that is desired) through multiple generations of cells. For example, it is intended that the term encompass many cell division cycles of the originally transfected cell. The term “stable transfection” or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the cell. The term “stable transfectant” refers to a cell that has stably integrated foreign DNA into the genomic DNA.
As used herein, the terms “transgene encoding,” “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides may, for example, determine the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus may code for the amino acid sequence.
As used herein, the term “wild type” (wt) refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants may be isolated, which are identified by the acquisition of altered characteristics when compared to the wild-type gene or gene product.
As used herein, the term “AAV virion,” “AAV particle,” or “AAV gene therapy particle,” “AAV gene therapy vector,” or “rAAV gene therapy vector” refers to a complete virus unit, such as a wt AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with at least one AAV capsid protein coat). In this regard, single-stranded AAV nucleic acid molecules of either complementary sense (e.g., “sense” or “antisense” strands) can be packaged into any one AAV virion and both strands are equally infectious. Also included are infectious viral particles containing a heterologous DNA molecule of interest (e.g., CFTR or a biologically active portion thereof), which is flanked on both sides by AAV ITRs.
As used herein, the term “transfection” refers to the uptake of foreign DNA by a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art (See e.g., Graham et al., Virol., 52:456 (1973); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier, (1986); and Chu et al., Gene 13:197 (1981). Such techniques may be used to introduce one or more exogenous DNA moieties, such as a gene transfer vector and other nucleic acid molecules, into suitable recipient cells.
As used herein, the terms “stable transfection” and “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term “stable transfectant” refers to a cell, which has stably integrated foreign DNA into the genomic DNA.
As used herein, the term “transient transfection” or “transiently transfected” refers to the introduction of foreign DNA into a cell wherein the foreign DNA fails to integrate into the genome of the transfected cell and is maintained as an episome. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term “transient transfectant” refers to cells which have taken up foreign DNA but have failed to integrate this DNA. As used herein, the term “transduction” denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via a replication-defective viral vector, such as via a recombinant AAV virion.
As used herein, the term “recipient cell” refers to a cell which has been transfected or transduced, or is capable of being transfected or transduced, by a nucleic acid construct or vector bearing a selected nucleotide sequence of interest. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected nucleotide sequence is present. The recipient cell may be the cells of a subject to which the gene therapy particles and/or gene therapy vector has been administered.
As used herein, the term “nucleic acid” sequence refers to a DNA or RNA sequence. Nucleic acids can, for example, be single or double stranded. The term includes sequences such as any of the known base analogues of DNA and RNA.
As used herein, the term “recombinant DNA molecule” refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
As used herein, the term “regulatory element” refers to a genetic element which can control the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
The term DNA “control sequences” refers collectively to regulatory elements such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need be present.
Transcriptional control signals in eukaryotes generally comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control sequences, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on the recipient cell type. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (See e.g., Voss et al., Trends Biochem. Sci., 11:287 (1986); and Maniatis et al., supra, for reviews). For example, the SV40 early gene enhancer is very active in a variety of cell types from many mammalian species and has been used to express proteins in a broad range of mammalian cells (Dijkema et al, EMBO J. 4:761 (1985)). Promoter and enhancer elements derived from the human elongation factor 1-alpha gene (Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene 91:217 (1990); and Mizushima and Nagata, Nucl. Acids. Res., 18:5322 (1990)), the long terminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. U.S.A. 79:6777 (1982)), and the human cytomegalovirus (Boshart et al., Cell 41:521 (1985)) are also of utility for expression of proteins in diverse mammalian cell types. Promoters and enhancers can be found naturally, alone or together. For example, retroviral long terminal repeats comprise both promoter and enhancer elements. Generally promoters and enhancers act independently of the gene being transcribed or translated. Thus, the enhancer and promoter used can be “endogenous,” “exogenous,” or “heterologous” with respect to the gene to which they are operably linked. An “endogenous” enhancer/promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer and promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
As used herein, the term “tissue specific” refers to regulatory elements or control sequences, such as a promoter, enhancers, etc., wherein the expression of the nucleic acid sequence is substantially greater in a specific cell type(s) or tissue(s). In particularly preferred embodiments, the CB promoter (CB is the same as CBA defined above) displays good expression of human CFTR, rAAV5-CB-.DELTA.264CFTR, rAAV5-CB-.DELTA.27-264CFTR, or another biologically active portion of CFTR. It is not intended, however, that the present invention be limited to the CB promoter or to lung specific expression, as other tissue specific regulatory elements, or regulatory elements that display altered gene expression patterns, are encompassed within the invention.
The presence of “splicing signals” on: an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.
Transcription termination signals are generally found downstream of a polyadenylation signal and are a few hundred nucleotides in length. The term “poly A site” or “poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be “heterologous” or “endogenous.” An endogenous poly A signal is one that is found naturally at the 3′ end of the coding region of a given gene in the genome. A heterologous poly A signal is one which has been isolated from one gene and operably linked to the 3′ end of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamHI/BclI restriction fragment and directs both termination and polyadenylation (Sambrook et al., supra, at 16.6-16.7).
The terms “operably linked” and “operatively linked” refer to the regulatory sequences for expression of the coding sequence that are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.
As used herein, the term “subject” refers to humans and other primates.
As defined herein, a “therapeutically effective amount” or “therapeutic effective dose” is an amount or dose of AAV particles or virions capable of producing sufficient amounts of a desired protein to restore the activity of the protein, thus providing a palliative tool for clinical intervention. A therapeutically effective amount or dose of transfected AAV particles that confer expression of a TREE (e.g., LEN), for example, to a patient will effect tissue regeneration.
The results presented herein demonstrate that:
These results are the first to (1) identify and validate the activity of tissue regeneration elements; and (2) demonstrate that enhancer element engineering can be used as a means to modulate tissue repair.
Accordingly, one aspect of the present disclosure provides a gene therapy construct comprising, consisting of, or consisting essentially of a nucleic acid encoding one or more tissue regeneration enhancer elements (TREEs) operatively linked to a promoter. In some embodiments, the gene therapy construct further comprises a nucleic acid sequence comprising one or more pro- or anti-regenerative factors, the expression of which is under the influence of the tissue regeneration enhancer elements.
In other embodiments, the gene therapy particle comprises a vector system. In some embodiments, the vector system comprises an AAV vector system.
In some embodiments, the gene therapy construct further comprises a first and second AAV inverted terminal repeat (ITR) sequence flanking the sequence encoding the TREEs. In certain embodiments, the ITR nucleotide sequences are derived from AAV serotype 2 (AAV-2).
Another aspect of the present disclosure provides a pharmaceutical composition comprising the gene therapy construct provided herein in a biocompatible pharmaceutical carrier.
In another embodiment, the TREE comprises the lepb-linked regulatory enhancer element (LEN).
One aspect of the present disclosure provides a method of treating or ameliorating tissue repair comprising, consisting of, or consisting essentially of administering to a subject a therapeutically effective amount of gene therapy construct as provided herein such that the tissue repair is treated or ameliorated.
Another aspect of the present disclosure provides a method of augmenting tissue regeneration in a subject comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a gene therapy construct as described herein such that the tissue regeneration is augmented.
Another aspect of the present disclosure provides a method of delivering cell therapy to a subject comprising, consisting of, or consisting essentially of inserting within the genome of reprogrammed stem/progenitor cells of the subject a gene therapy construct as provided herein such that cell therapy is delivered.
Another aspect of the present disclosure provides a method for screening drugs that modulate regenerative capacity comprising, consisting of, or consisting essentially of transfecting a gene construct as described herein into a model system, administering to the system a drug of interest, and measuring regenerative capacity in response to the drug.
In some embodiments, the model system comprises a zebrafish model system.
Another aspect of the present disclosure provides all that is disclosed and illustrated herein.
The following examples are provided as illustration and not by way of limitation.
Wild-type or transgenic male and female zebrafish of the outbred Ekkwill (EK) strain were used for all experiments, with adults ranging in age from 3 to 12 months. Water temperature was maintained at 26° C. for animals unless otherwise indicated. Fins were amputated to 50% of their original length using razor blades. As penetrance of the dob mutation was higher at 33° C. than at 26° C., dob fish were maintained at 33° C. after caudal fin amputation. To measure lengths of regenerates, lengths from the amputation plane to the distal tips of the 3rd and 4th fin rays of dorsal and ventral caudal fin lobes were determined using ZEN software. Because some dob animals regenerated portions of the 1st and 2nd fin rays of ventral lobes, regenerating caudal fin areas for
To generate lepb:eGFP BAC transgenic animals (full names, Tg(lepb:eGFP)pd120 and Tg(lepb:eGFP)pd121), the iTol2 cassette41 was integrated into the BAC clone DKEY-21022 using Red/ET recombineering technology (GeneBridges). Then, the first exon of the lepb gene in the BAC clone DKEY-21022 was replaced with an eGFP cassette by Red/ET recombineering. 5′ and 3′ homology arms were amplified by PCR (Supplementary Information) and subcloned into the pCS2-eGFP plasmid. One nl of 50 ng/μl purified, recombined BAC was injected into one-cell stage zebrafish embryos along with one nl of 30 ng/μl synthetic Tol2 mRNA41. To sort F0 transgenic animals injected with lepb:eGFP constructs, fin folds were amputated at 3 or 4 dpf, and embryos displaying eGFP fluorescence near the injury site at 1 dpa were selected, (
To define LEN activity, over 60 additional new transgenic lines were established in this study, listed in Table 1. To generate transgenic animals, DNA sequences were amplified by PCR with indicated primers (Table 2) and subcloned into a pCS2-eGFP-1-scel vector, in which 1-Scel restriction sites were flanked by a multiple cloning site. As promoters, 2 kb, 1.6 kb, and 0.7 kb upstream sequences of lepb, cmlc242 and α-cry43 genes were used, respectively. These constructs were injected into one-cell-stage wild-type or dob embryos using standard meganuclease transgenesis techniques. 2 kb lepb upstream sequences could induce transgene expression after fin fold amputation at larval stages, but never alter caudal fin amputation in adults. To isolate stable lines, larvae were examined for transgene expression near injury site in response to fin fold amputation (2 kb lepb), in cardiomyocytes (1.6 kb cmlc2), and in lens (0.7 kb α-cry).
To test additional TREES, we subcloned putative enhancer regions of i111a, plek, vcana, and cd248b upstream of 800 bp of lepb upstream sequence (P0.8). To define TREE activity, these constructs were injected into one-cell-stage wild-type embryos, Fin folds were amputated at 4 dpf, and eGFP fluorescence near the amputation plane was examined at 5 dpf (1 dpa).
Transgenic mice (CD-1 strain) were generated by oocyte microinjection as described previously.44 LEN-hsp68::lacZ transgenic mice were generated by subcloning the zebrafish LEN enhancer sequence into the transgenic reporter plasmid hsp684acZ45. Ctrl-hsp68::lacZ transgenic mice harbor a transgene, Prkaa2[mMEF2(1+2)]-hsp68-lacZ, which contains a modified version of a 931-bp enhancer sequence from the mouse Prkaa2 gene cloned into hsp68-lacZ (J. Hu and B. L. Black, unpublished observations). Partial apical resection injury in male and female neonatal mice at postnatal day 1 was performed similarly to previously described methods46. Hearts and paws were collected at postnatal day 4. All experiments with mice complied with federal and institutional guidelines and were reviewed and approved by the UCSF IACUC.
RNA was isolated from dissected caudal tins and partially resected ventricles using Tri-Reagent (Sigma). cDNA was synthesized from 1 μg of total RNA using the Roche First Strand Synthesis Kit. Quantitative PCR was performed using the Roche LightCycler 480 and the Roche LightCycler 480 Probes Master. All samples were analyzed in biological triplicates and technical duplicates. Primer sequences are given in Table 2. Probe numbers for actb2, lepb, and nrg1 were 104, 156 and 76, respectively. lepb and nrg1 transcript levels were normalized to actb2 levels for all experiments.
Total RNA was prepared from two biological replicate pools of ablated Z-CAT ventricles and uninjured ventricles at 7 days post-ablation as per Gemberling et al.31, or regenerating and uninjured caudal fins. Generation of mRNA libraries and sequencing were performed at the Duke Genome Sequencing Shared Resource using an Illumina HiSeq2000. Sequences were aligned to the zebrafish genome (Zv9) using TopHat47. Differentially regulated transcripts were identified using EdgeR and an FDR cut-off of O.148 Accession numbers for transcriptome datasets are GSE75894 and GSE76564.
To identify candidate enhancer elements activated during heart regeneration, chromatin extracts were prepared from two biological replicate pools of 10 ablated Z-CAT ventricles and 10 uninjured ventricles. Chromatin was sonicated and immunoprecipitated with an antibody against H3K27ac (ActiveMotif) using the MAGnify ChIP system (Invitrogen). Sequencing libraries were prepared as per Bowman, et al.49. Sequencing was performed using an Illumina HiSeq2000, and 10-25 million 50 bp single end reads were obtained for each library. Sequences were aligned to the zebrafish genome (Zv9) using Bowtie250. Differential peaks were identified using Model-based Analysis for ChiP-Seq (MACS)51.
In situ hybridization on cryosections of 4% paraformaldehyde-fixed fins was performed as described previously52. To generate digoxigenin-labeled probes for lepb and fgf20a, we generated a fragment of lepb cDNA and a full length of fgf20a cDNA by PCR using primer sequences described in Table 2. The nrg1 probe was prepared as described previously31. Immunohistochemistry was performed as described previously40. Primary and secondary antibodies used in this study were: anti-Myosin heavy chain (mouse, F59, Developmental Studies Hybridoma Bank), anti-MEF2 (rabbit, sc-313, Santa Cruz Biotechnology), anti-PCNA (mouse, P8825, Sigma), anti-eGFP (rabbit, A11122, Life Technologies), anti-eGFP (chicken, GFP-1020. Ayes Labs), anti-Raldh2 (rabbit, Abmart), anti-Ds-Red (rabbit, 632496, Clontech), anti-p63 (mouse; 4A4, Santa Cruz Biotechnology), Alexa Fluor 488 (mouse and rabbit; Life Technologies), Alexa Fluor 594 (mouse and rabbit; Life Technologies). For EdU incorporation experiments, zebrafish were injected intraperitoneally with 10 mM EdU (A10055, sigma), and caudal fins were collected at 1 hour post-treatment. EdU staining was performed as previously described53. The secondary antibody used for EdU staining was Alexa 488 azide (10-20 μM, Sigma). Whole-mount images were acquired using an M205FA stereofluorescence microscope (Leica) or Axio Zoom (Zeiss). Images of tissue sections (10 μm for hearts and 14 Am for fins) were acquired using an LSM 700 confocal microscope (Zeiss). X-gal staining to detect β-galactosidase activity and counterstaining with nuclear fast red were performed with murine tissue as described previously44.
Clutchmates were randomized into different treatment groups for each experiment. No animal or sample was excluded from the analysis unless the animal died during the procedure. Sample sizes were chosen on the basis of previous publications and experiment types, and are indicated in each figure legend or methods. For expression patterns, at least five fish from each transgenic line were examined. At least 9 hearts of each group were pooled for RNA purification and subsequent RT-qPCR. Quantification of cardiomyocyte proliferation and calculation of statistical outcomes were assessed by a person blinded to the treatments. Sample sizes, statistical tests, and P values are indicated in the figures or the legends. One-way ANOVA tests were applied when normality and equal variance tests were passed. The Mann-Whitney rank sum test was applied in assays of cardiomyocyte proliferation.
How tissue regeneration programs are triggered by injury has received limited research attention. Here, we investigated the existence of enhancer regulatory elements that engage in regenerating tissue. Transcriptome analyses revealed that leptin b (lepb) is sharply induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for tissue regeneration enhancer elements (TREES) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs.
The capacity for complex tissue regeneration is unevenly distributed among vertebrate tissues and species. Salamanders and zebrafish possess remarkable potential to regenerate tissues like amputated appendages, resected heart muscle, and transected spinal cords1,2. Investigations of gene expression arid function have generated molecular models for regeneration in multiple contexts, yet there is a deficiency in our understanding of the regulatory events that activate tissue regeneration programs1-5.
Recent genome-wide chromatin analyses suggest that gene regulatory elements comprise a substantial portion of genomic sequence. Of these elements, distal-acting regulatory sequences, or enhancers, represent the most abundant class6,7. Enhancers can direct expression of their target genes and have been predominantly examined as a means for stage- and tissue-specific regulation during embryonic development8,9. Studies have also implicated enhancers in disease and as targets during evolution10-15. Because of such findings, it is possible there may also exist enhancer elements that engage in response to tissue damage to regulate genetic programs for regeneration. The identification of such elements could potentially inspire solutions for manipulating regenerative events.
To identify genes that are induced during tissue regeneration, we collected RNA from uninjured and regenerating tissues of adult zebrafish and sequenced transcriptomes. Our analyses identified 2,408 genes with significantly higher expression in tail fins at 4 days post amputation (dpa), and 859 genes with significantly higher expression in cardiac ventricles 7 days after induced genetic ablation of half of all cardiomyocytes (
To capture the regulatory elements responsible for lepb induction, we replaced the first exon of lepb with an eGFP reporter transgene within a 150 kb BAC containing 105 kb of DNA sequence upstream of the start codon (
Enhancers are identifiable as areas of open chromatin, bound by transcription factors and occupied by histones possessing various modifications, such as acetylated lysine 27 of Histone H3 (H3K27ac)19,20. To define areas of open chromatin, we assayed genomic regions surrounding lepb for H3K27ac marks by ChIP-Seq in samples of uninjured and regenerating hearts. Two regions within the lepb BAC, located 7 kb and 3 kb upstream of the lepb start codon, were enriched with H3K27ac marks in regenerating, but not uninjured, samples (
We next examined whether an isolated 1.3 Kb sequence that corresponded to the H3K27ac-rich region could activate gene expression when fused to P2, which ostensibly includes the lepb promoter (
Analysis of regions upstream of Leptin genes in murine and human genomes revealed limited primary sequence conservation of LEN (
To identify minimal sequences responsible for the activity of LEN, we tested the ability of various fragments to direct regeneration-activated expression. We found that more distal LEN fragments composed of nucleotides 1-850, 450-1000, 450-850, or 660-850 could each drive eGFP expression from the lepb 2 kb promoter during fin regeneration (
We analyzed sequences of the minimal 190 nt (fin) and 316 nt (heart) elements, and identified distinct sets of predicted transcription factor binding motifs. LEN(663-854) contains predicted AP-1, Sox, Forkhead, and ETS binding sites, and we confirmed by transgenic reporter assays that a predicted AP-1 binding site at LEN(776-782) is necessary to direct expression in regenerating fins (
Recent studies have described new enhancer-target gene pairings caused by chromosomal rearrangements that underlie genetic diseases like cancer and neurological disorders10,12,15. To examine a parallel idea for experimentally guiding tissue regeneration, we designed transgenic constructs positioning LEN and the minimal lepb promoter upstream of pro- or anti-regenerative factors. A possible outcome is that LEN would limit embryonic expression of potent developmental influences to permit maturation from the one-cell stage to adulthood, but also trigger and sustain expression of these influences upon tissue damage.
To create enhancer-effector transgenes, we took advantage of the dependency of fin regeneration on signaling by Fibroblast growth factors (Fgfs)4,27. We first positioned LEN upstream of a cDNA encoding a dominant-negative form of fgfr1 (dnfgfr1)—a potent inhibitor of embryonic development27,28—and injected this construct into wild-type embryos. We established stable lines of zebrafish harboring either P2:dnfgfr1 or LENP2:dnfgfr1, demonstrating that dnfgfr1 expression was limited to developmentally insignificant levels. Adult P2:dnfgfr1 fins displayed no detectable dnfgfr1 induction after amputation and regenerated normally. By contrast, injury to LENP2:dnfgfr1 animals induced strong expression of dnfgfr1 (detectable by dnfgfrl-eGFP fusion protein fluorescence) that was restricted to the amputation plane. Moreover, these animals displayed conspicuous defects or outright failures in fin regeneration (
We complemented these experiments with a gain-of-function approach, based on the discovery that mutations in the fgf20a ligand gene, devoid of blastema (dob), arrest fin regeneration4. We positioned LEN and the minimal lepb promoter upstream of a fgf20a cDNA and injected this construct into one-cell dob embryos. We generated stable lines of control dob; P2:fgf20a and dob; LENP2:fgf20a animals, indicating that these constructs restricted ectopic fgf20a expression during embryonic development. Upon amputation of adult tail fins, dob; P2:fgf20a animals induced no additional detectable fgf20a and displayed regenerative blocks comparable to dob animals (
Heart regeneration occurs through injury-induced stimulation of proliferation by pre-existing cardiomyocytes29. Recent evidence indicates that the secreted factor Neuregulin1 (Nrg1) is a cardiomyocyte mitogen during cardiac growth or repair in lower and higher vertebrates30-32. In zebrafish, nrg1 is present at very low levels in the heart, and it is induced upon injury at levels that remain undetectable by standard ISH methodology31. Strong transgenic overexpression of nrg1 in adult zebrafish cardiomyocytes activates overt cardiomyocyte proliferation and enlarges the ventricular wall31. To test whether LEN can influence heart regeneration, we created stable transgenic zebrafish lines with P2:nrg1 or LENP2nrg1 constructs. Resection of the ventricular apex sharply increased nrg1 transcripts in injured portions of LENP2:nrg but not control P2:nrg1, ventricles (
Here, we used a profiling approach to identify small regulatory elements that direct gene expression in regenerating tissue, which we now refer to as Tissue Regeneration Enhancer Elements (TREES). Recently, a ˜18 Kb region of the murine Bmp5 locus was reported to activate expression from minimal promoters in injury contexts33, suggesting it may harbor a TREE analogous to the LEN element we describe here. We suspect that diverse classes of TREES exist, including elements activated during development and re-activated by injury34 or during regeneration, elements that activate expression preferentially during regeneration in multiple tissues, and regeneration-specific elements that are more tissue-restricted. The investigation of individual binding motifs within TREES should identify upstream transcriptional regulators of regeneration, whereas genomic TREE locations can pinpoint novel downstream target genes.
Current methodologies to interrogate regenerative biology often have experimental disadvantages like multiple transgenes, ubiquitous promoters, irreversible expression, and/or stressful stimuli like estrogen analogs, tetracycline analogs, or heat shock35. By contrast, TREES are single-transgene systems that can naturally induce and maintain target genes upon injury, and then naturally temper expression as regeneration concludes. Whereas LEN elements induce expression in fin mesenchyme and/or endocardium, we expect that future investigations will uncover a panel of regeneration-responsive TREEs representing additional distinct tissues. Thus, when combined with effectors, recombinases, or genome-editing enzymes, TREEs should facilitate targeted genetic manipulations that have been elusive to this point.
Multiple features of TREEs are appealing with respect to the design of potential regenerative therapies. Previous studies have implicated the manipulation of enhancer activity as a means to treat human genetic disease12,36. In this study, we report that pro-or anti-regenerative factors directed by TREEs are capable of blocking regenerative growth, promoting cell proliferation, or even rescuing genetic defects in regeneration. With a TREE-based system, factor delivery is spatiotemporally defined and could permit therapeutic cycles as injury recurs. Notably, although Nrg1 impacts heart regeneration, systemic neuregulin delivery has the potential for neurological or oncogenic effects37,38. Thus, enhancer-based targeting of Nrg1 to injury sites, as we model here in zebrafish, may represent a more effective regenerative medicine platform. We suggest that TREES identified from natural regenerative contexts across vertebrate species can inform new strategies for precise factor delivery to injured human tissues.
To test whether peaks in our profiling datasets represented additional enhancer elements similar to LEN, we performed F0 transgenic analysis. We used 800 nt of upstream sequence of lepb (P0.8) as a minimal promoter, as embryos injected with P0.8:EGFP only very rarely displayed EGFP fluorescence during fin fold regeneration (
To test enhancer activity during regeneration, we first analyzed EGFP transgene induction in F0 mosaic transgenic animals. We fused LEN fragments to the lepb 2 kb upstream sequence (P2). After microinjection of LENP2 constructs, potential founders were selected at the larval (5 dpf) stage using an assay for induced EGFP fluorescence in response to fin fold amputation. At 60-90 dpf, caudal fins were amputated and EGFP fluorescence was examined (
To examine whether LEN fragments are functional with different promoters, we tested enhancer activity with cmlc2 and α-cry promoters. A total of 21% (8/38), 21% (6/28), 40% (16/40), and 20% (1/5) of F0 mosaic transgenic adult fish containing (1-1350), (1-850), (450-1000), and (660-850) LEN fragments coupled with the cmlc2 promoter, respectively, displayed enhancer activity during fin regeneration (
To test whether the LEN element can be engineered to modulate tissue regeneration, we positioned LEN in constructs with a dominant-negative fgfr1, a potent inhibitor of fin regeneration. P2:dnfgfr1-EGFP and LENP2:dnfgfr1-EGFP constructs contained an ef1α-nls-mCherry mini-gene embedded in same vector, used as a screening marker. We injected these constructs into wild-type embryos, selected transgene-positive (red) embryos, and examined fin regeneration at 3 months of age. Whereas all 27 P2:dnfgfr1 F0 transgenic fish regenerated fins properly, 7 of 67 LENP2:dnfgfr1 F0 transgenic fish showed defective regeneration of some fin rays, corresponding to where LEN ectopically induced dnfgfr1-EGFP (
To examine whether a LEN element coupled with the Fgf ligand gene fgf20a could rescue the defective fin regeneration in dob mutants, we injected two constructs into dob embryos: 1) fgf20a downstream of the lepb 2 kb promoter, with ef1α-nls-mCherry as a screening marker (dob; P2:fgf20a); and 2) this same construct with a LEN sequence positioned upstream of P2 (dob; LENP2:fgf20a). We examined fin regeneration in transgene-positive (red) F0 mosaic adult transgenic fish. Whereas 2 of 19 dob, and 2 of 40 dob; P2:fgf20a F0 animals regenerated more than 500,000 μm2 of tissue at 10 dpa, 11 of 44 dob; LENP2:fgf20a F0 animals regenerated more than 500,000 μm2 (
To determine whether enhancer elements related to LEN are found near genes that show significantly higher levels during regeneration than in uninjured tissues, we performed BLAST using fin and endocardial LEN elements. Blast analysis revealed genomic regions that show homology with LEN near some genes, listed in Tables 4 and 5 below.
8. Analysis of lepr:lepr-mCherry Reporter and lepb Mutant Lines.
To visualize which cells might respond to Lepb during tissue regeneration, we created a lepr BAC reporter line. To generate lepr:lepr-mCherry BAC transgenic animals (formal name Tg(lepr:lepr-mCherry)pd95), the iTol2 cassette was integrated into the BAC clone DKEY-1K24. Then, a mCherry cassette was integrated at the C-terminus of Lepr, which results in a C-terminal mCherry-fusion of Lepr. (
To examine requirements for lepb in zebrafish tissue regeneration, we used TALE Nucleases to generate mutations in the a-helix C of Lepb (
It will be readily apparent to one of ordinary skill in the relevant arts that suitable modifications and adaptations to the compositions, methods, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of the claimed embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in all variations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof as noted, unless other specific statements of incorporation are specifically provided.
This application claims priority to U.S. Provisional Patent Application No. 62/309,649, filed Mar. 17, 2016, the entirety of which is hereby incorporated by reference.
This invention was made with Government support under Federal Grant No. R01 GM074057. The Federal Government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62309649 | Mar 2016 | US |
