Patent Application
20240299347
All patents, patent applications and publications, and other literature references cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by progressive cognitive, behavioral, motor dysfunctions and short life-spans. HD is caused by a CAG trinucleotide repeat expansion in Exon 1 of the HTT gene, which results in an expanded polyglutamine (polyQ) tract in the encoded huntingtin protein, referred to as mutant huntingtin (mHTT). The mHTT exhibits toxic gain of functions causing neuronal dysfunction and cell death. The proteolytic cleavage of mHTT is a key event in the molecular pathogenesis of HD and it is believed that the N-terminal fragment containing the expanded polyglutamine of mHTT plays an important role in the pathogenesis of HD. Although the genetic cause of HD is well established, the cellular and molecular mechanisms involved in mHTT-mediated early neuronal dysfunction and late neurodegeneration are not fully understood.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising: (i) identifying the subject as expressing of mutant huntingtin (mHTT), and (ii) administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising: (i) identifying the subject as having an increased expression level of arachidonate 5-lipoxygenase (ALOX5), and (ii) administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising: (i) identifying the subject as having an increased expression level of 5-lipoxygenase-activating protein (FLAP), and (ii) administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In some embodiments, the subject expresses mHTT, an increased level of ALOX5, an increased level of FLAP, or a combination thereof. In some embodiments, expression of mHTT is determined from a sample from the subject. In some embodiments, expression level of ALOX5 or FLAP is determined from a sample from the subject. In some embodiments, expression or expression level is a protein level, a mRNA expression level, or combination thereof.
In some embodiments, the subject further expresses increased level of ALOX5, an increased level of FLAP, or a combination thereof. In some embodiments, the subject further expresses mHTT, an increased level of FLAP, or a combination thereof. In some embodiments, the subject further expresses mHTT, or an increased level of ALOX5 or a combination thereof.
In some embodiments, the composition reduces ALOX5 expression in the subject compared to ALOX5 expression in a subject suffering from Huntington's disease or compared to ALOX5 expression in the subject before administration of the composition. In some embodiments, the composition reduces FLAP expression in the subject compared to FLAP expression in a subject suffering from Huntington's disease or compared to FLAP expression before administration of the composition. In some embodiments, the composition inhibits or reduces ALOX5 expression in the subject. In some embodiments, the composition inhibits or reduces FLAP expression in the subject. In some embodiments, the composition comprises Zileuton. In some embodiments, the composition comprises MK.886. In some embodiments, the composition comprises docebenone (AA 861). In some embodiments, the composition comprises boswellic acids. Insome embodiments, the composition comprises atreleuton (ABT-761 or VIA-2291). In some embodiments, the composition comprises setileuton (1,3,4-oxadiazole MK-0633). In some embodiments, the composition comprises PF-4191834 or CJ-13610. In some embodiments, the composition comprises Flavocoxid.
In some embodiments, the composition comprises a ALOX5 small interfering ribonucleic acid (siRNA). In some embodiments, the composition comprises a FLAP small interfering ribonucleic acid (siRNA).
In some embodiments, the composition comprises an ALOX5 short-hairpin ribonucleic acid (shALOX5). In some embodiments, the composition comprises a FLAP short-hairpin ribonucleic acid (shFLAP).
In some embodiments, the composition comprises a guide RNA or a single-molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5. In some embodiments, the composition comprises a guide RNA or a single-molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the guide RNA or the single molecule guide RNA is pre-complexed with a DNA endonuclease. In some embodiments, the DNA endonuclease is a Cas9 or dCas9 endonuclease.
In some embodiments, the composition comprises a vector. In some embodiments, the composition comprises a viral vector, wherein the viral vector encapsulates a nucleic acid encoding the shALOX5, a nucleic acid encoding the shFLAP, a nucleic acid encoding the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5, or a nucleic acid encoding the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the viral vector is an adeno-associated vector (AAV).
In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the composition is delivered systemically.
In some embodiments, the human subject has an increased expression level of ALOX5 compared to a subject not suffering from Huntington's disease. In some embodiments, the human subject has an increased expression level of FLAP compared to a subject not suffering from Huntington's disease.
In certain aspects, described herein is a composition for treating or preventing Huntington's disease, comprising a composition targeting the ACSL4-independent ferroptosis pathway. In some embodiments, the composition reduces arachidonate 5-lipoxygenase (ALOX5) expression in a subject in need thereof. In some embodiments, the composition reduces 5-lipoxygenase-activating protein (FLAP) expression in a subject in need thereof.
In some embodiments, the composition comprises Zileuton. In some embodiments, the composition comprises MK.886. In some embodiments, the composition comprises docebenone (AA 861). In some embodiments, the composition comprises boswellic acids. Insome embodiments, the composition comprises atreleuton (ABT-761 or VIA-2291). In some embodiments, the composition comprises setileuton (1,3,4-oxadiazole MK-0633). In some embodiments, the composition comprises PF-4191834 or CJ-13610. In some embodiments, the composition comprises Flavocoxid. In some embodiments, the composition comprises an ALOX5 small interfering ribonucleic acid (siALOX5). In some embodiments, the composition comprises a FLAP small interfering ribonucleic acid (siFLAP). In some embodiments, the composition comprises an ALOX5 short hairpin ribonucleic acid (shALOX5). In some embodiments, the composition comprises a FLAP short hairpin ribonucleic acid (shFLAP). In some embodiments, the composition comprises a guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5. In some embodiments, the composition comprises a guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the guide RNA or the single molecule guide RNA is pre-complexed with a DNA endonuclease. In some embodiments, the DNA endonuclease is a Cas9 or dCas9 endonuclease.
In some embodiments, the composition further comprises a viral vector, wherein the viral vector encapsulates a nucleic acid encoding the shALOX5, the shFLAP, the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5, or the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the viral vector is an AAV vector.
In certain aspects, described herein is small interfering ribonucleic acid (siRNA) targeting the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway. In some embodiments, the siRNA is an ALOX5 small interfering ribonucleic acid (siALOX5). In some embodiments, the siRNA is a FLAP small interfering ribonucleic acid (siFLAP). In certain aspects, described herein is a vector comprising a SiRNA.
In certain aspects, described herein is a nucleic acid comprising a sequence encoding a short hairpin ribonucleic acid (shRNA) targeting the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway. In certain aspects, described herein is a vector comprising any of the nucleic acid described above. In certain aspects, described herein is a viral vector comprising the nucleic acid described above. In some embodiments, the viral vector is an AAV vector.
In certain aspects, described herein is a method of diagnosing Huntington's disease in a subject comprising: (i) determining the presence of mutant huntingtin (mHTT) in the subject. In certain aspects, described herein is a method of diagnosing Huntington's disease in a subject comprising: (i) determining the level of arachidonate 5-lipoxygenase (ALOX5) in the subject; and (ii) diagnosing the subject with Huntington's disease if the level of ALOX5 expression in the subject is increased as compared to a subject not suffering from Huntington's disease. In some embodiments, the method further comprises determining the level of FLAP in the subject. In some embodiments, the method further comprises determining the whether the subject expresses mHTT. In certain aspects, described herein is a method of diagnosing Huntington's disease in a subject comprising: (i) determining the level of 5-lipoxygenase-activating protein (FLAP) in the subject; and (ii) diagnosing the subject with Huntington's disease if the level of FLAP expression in the subject is increased as compared to a subject not suffering from Huntington's disease. In some embodiments, the method further comprises determining the level of ALOX5 in the subject. In some embodiments, the method further comprises determining whether the subject expresses mHTT. In some embodiments the level of ALOX5, FLAP, and/or mHTT is determined from a sample from the subject. In some embodiments, the subject is diagnosed with Huntington's disease if the level of both FLAP and ALOX5 expression in the subject is increased as compared to a subject not suffering from Huntington's disease. In some embodiments, the subject is diagnosed with Huntington's disease if the level of FLAP expression in the subject is increased as compared to a subject not suffering from Huntington's disease and the subject expresses mHTT. In some embodiments, the subject is diagnosed with Huntington's disease if the level of ALOX5 expression in the subject is increased as compared to a subject not suffering from Huntington's disease and the subject expresses mHTT. In some embodiments, the subject is diagnosed with Huntington's disease if the level of both ALOX5 and FLAP expression in the subject is increased as compared to a subject not suffering from Huntington's disease and the subject expresses mHTT. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.
The patent or application file contains at least one drawing executed in color.
All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present disclosed subject matter pertains.
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 invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
As used herein, the term “subject” refers to a vertebrate animal. In one embodiment, the subject is a mammal or a mammalian species. In one embodiment, the subject is a human. In one embodiment, the subject is a healthy human adult. In other embodiments, the subject is a non-human vertebrate animal, including, without limitation, non-human primates, laboratory animals, livestock, racehorses, domesticated animals, and non-domesticated animals. In one embodiment, the term “human subjects” means a population of healthy human adults.
The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.
As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
As used herein the term “variant” covers nucleotide or amino acid sequence variants which have about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, or about 65% nucleotide identity, or about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, or about 65% amino acid identity, including but not limited to variants comprising conservative, or non-conservative substitutions, deletions, insertions, duplications, or any other modification. The term variant as used herein includes functional and non-functional variants, and variants with reduced or altered activity.
The present disclosure provides compositions and methods for treating and/or preventing Huntington's disease (HD). Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Described herein is the discovery that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mouse.
Described herein is the discovery that ALOX5 mediates the ACSL4-independent ferroptosis induced by HTTQ94 via the stabilization of FLAP. By using RNAi-mediated screening, ALOX5 was identified as a major factor required for the ACSLA-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a promising new target for Huntington's disease. Moreover, mHTT-mediated ferroptosis is effectively blocked by Zileuton, a specific inhibitor of ALOX5. Silencing RNA (siRNA) can downregulate gene expression of specific genes (Agrawal N, Dasaradhi PVN, Mohmmed A, Malhotra P, Bhatnagar R K, Mukherjee S K. RNA Interference: Biology, Mechanism, and Applications. Microbial Mol Biol Rev. 2003 Dcc; 67(4): pp. 657-685).
As used herein mHTT refers to polyglutamine-expanded mutant huntingtin. Huntington's disease is caused by a CAG trinucleotide repeat expansion in exon 1 of the HTT gene, which results in an expanded polyglutamine (polyQ) tract in the encoded huntingtin protein, referred to as mutant huntingtin (mHTT).
As used herein HTTQ94 refers to the terminal fragment containing the expanded polyglutami. It is the mHTT fragment with the expanded 94 glutamine residues.
As used herein ALOX5 refers to arachidonate 5-lipoxygenase. Information on ALOX5 is available at www.ncbi.nlm.nih.gov/gene/240, the content of which is hereby incorporated by reference in its entirety include the nucleic acid and amino acid sequences of ALOX5.
As used herein FLAP refers to arachidonate 5-lipoxygenase activating protein. Information on FLAP is available at www.ncbi.nlm.nih.gov/gene/241, the content of which is hereby incorporated by reference in its entirety include the nucleic acid and amino acid sequences of FLAP.
In certain aspects, described herein is the use of small interfering RNA (siRNA) platforms to silence the arachidonate 5-lipoxygenase (ALOX5) or 5-lipoxygenase-activating protein (FLAP). Also described herein is the use of ALOX5, FLAP, or both as a biomarker for diagnosing Huntington's disease.
The disclosure further provides methods for treating and/or preventing Huntington's disease in patients possessing increased expression of ALOX5, FLAP, or both. The compositions and methods disclosed herein can be used as disease modifying therapies to enable prevention or treatment of Huntington's disease and related disorders earlier in disease progression and improve clinical outcomes. The disclosure is based, at least in part, on the discovery, that ALOX5 expression is essential for HTTQ94-mediated ferroptosis, and that HTTQ94 promotes ALOX5-mediated ferroptosis by interacting with FLAP and up-regulating its stability. Described herein are methods of treatment or preventing Huntington's disease comprising either silencing or reducing expression of ALOX5, FLAP, or both among subjects who have high ALOX5 or FLAP expression compared to a subject who is not suffering from Huntington's disease.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising: (i) identifying the subject as having an expression of mutant huntingtin (mHTT), and (ii) administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising: (i) identifying the subject as having an increased expression level of arachidonate 5-lipoxygenase (ALOX5), and (ii) administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In certain aspects, described herein is a method of treating or preventing Huntington's disease in a subject in need thereof, comprising: (i) identifying the subject as having an increased expression level of 5-lipoxygenase-activating protein (FLAP), and (ii) administering to said subject a composition that targets the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway.
In some embodiments, the subject expresses mHTT, an increased level of ALOX5, or an increased level of FLAP or a combination thereof. In some embodiments, expression of mHTT is determined from a sample from the subject. In some embodiments, expression level of ALOX5 and/or FLAP is determined from a sample from the subject. In some embodiments, expression or expression level is a protein level, a mRNA expression level, or combination thereof. Methods for detecting and quantifying ALOX5 or FLAP mRNA in biological samples are known the art. In some embodiments, the level of mRNA is determined using RT-qPCR or RNA-seq.
In one embodiment, a sample comprises, a blood sample, serum, cells (including whole cells, cell fractions, cell extracts, and cultured cells or cell lines), tissues (including tissues obtained by biopsy), body fluids (e.g., urine, sputum, amniotic fluid, synovial fluid), or from media (from cultured cells or cell lines). In some embodiments, the sample is blood or blood serum or CSF. In some embodiments, the same is cells of the subject. In some embodiments, the sample is nervous system tissue of the subject. In some embodiments, the sample comprises brain cells of the subject.
In some embodiments, the subject expressing mHTT further expresses increased level of ALOX5, or an increased level of FLAP or a combination thereof. In some embodiments, the subject expressing an increased level of ALOX5 further expresses mHTT, or an increased level of FLAP or a combination thereof. In some embodiments, the subject expressing an increased level of FLAP further expresses mHTT, or an increased level of ALOX5 or a combination thereof. In some embodiments, the increased levels are ALOX5 mRNA levels, ALOX5 protein levels, or both ALOX5 mRNA levels and ALOX5 protein levels in a sample from the subject. In some embodiments, the increased levels are FLAP mRNA levels, FLAP protein levels, or both FLAP mRNA levels and FLAP protein levels in a sample from the subject. In some embodiments, ALOX5 or FLAP mRNA levels and ALOX5 or FLAP protein levels the from a sample from the subject.
In some embodiments, the composition inhibits or reduces ALOX5 expression in the subject. In some embodiments, the composition inhibits or reduces FLAP expression in the subject.
In some embodiments, the composition reduces ALOX5 expression in the subject compared to ALOX5 expression in a subject suffering from Huntington's disease or compared to ALOX5 expression in the subject before administration of the composition. ALOX5 expression may be reduced using any known method in the art. The expression of ALOX5 may be reduced by at least about 5% to about 95%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to corresponding expression of ALOX5 in a subject suffering from Huntington's disease or in the subject before administration of the composition. The expression of ALOX5 may be silenced relative to corresponding expression of ALOX5 in a subject suffering from Huntington's disease or in the subject before administration of the composition. In some embodiments, the expression of ALOX5 is reduced cells of the subject.
In some embodiments, the composition reduces FLAP expression in the subject compared to FLAP expression in a subject suffering from Huntington's disease or compared to FLAP expression before administration of the composition. FLAP expression may be reduced using any known method in the art. In some embodiments, the composition inhibits or reduces ALOX5 expression in the subject. In some embodiments, the composition inhibits or reduces FLAP expression in the subject. The expression of FLAP may be reduced by at least about 5% to about 95%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to corresponding expression of FLAP in a subject suffering from Huntington's disease or in the subject before administration of the composition. The expression of FLAP may be silenced relative to corresponding expression of FLAP in a subject suffering from Huntington's disease or in the subject before administration of the composition. In some embodiments, the expression of FLAP is reduced cells of the subject.
In some embodiments, the composition comprises Zileuton. Zileuton is an FDA-approved orally active inhibitor of ALOX5 used for the maintenance treatment of asthma. Zileuton has the chemical name (+)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea and the chemical structure below. In some embodiments, the composition comprises MK.886. MK.886 is a cell-permeable and orally active FLAP Inhibitor. MK.886 has the chemical structure below.
In some embodiments, the composition comprises docebenone (AA 861). is a potent, selective and orally active 5-LO (5-lipoxygenase) inhibitor. In some embodiments, the composition comprises
In some embodiments, the composition comprises boswellic acids. 11-keto-boswellic acids inhibit ALOX5. In some embodiments, the composition comprises atreleuton (ABT-761 or VIA-2291). Atreleuton is a selective, reversible, and orally bioavailable ALOX5 inhibitor. In some embodiments, the composition comprises
In some embodiments, the composition comprises setileuton (1,3,4-oxadiazole MK-0633). Setileuton (1,3,4-oxadiazole MK-0633), a potent and selective ALOX5 inhibitor. In some embodiments, the composition comprises
In some embodiments, the composition comprises PF-4191834 or CJ-13610. In some embodiments, the composition comprises
In some embodiments, the composition comprises flavocoxid. Flavocoxid is a mixture of purified bioflavonoids that inhibits COX-1, COX-2, and ALOX5 in vitro and in animal models.
In some embodiments, the composition comprises a ALOX5 small interfering ribonucleic acid (siALOX5). For example, in various embodiments the composition is a vector comprising at least one of the small interfering ribonucleic acid (siRNA) designs targeting ALOX5. In various embodiments, the siALOX5 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the siRNA designs of ALOX5. In some embodiments, the composition comprises a FLAP small interfering ribonucleic acid (siFLAP). For example, in various embodiments the composition is a vector comprising at least one of the small interfering ribonucleic acid (siRNA) designs targeting FLAP. In various embodiments, the siRNA is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the siRNA designs of FLAP. In some embodiments, the siRNA comprises one or more gene-specific siRNAs (e.g. siRNA targeting ALOX5 or FLAP) as a pooled siRNA. In some embodiments, the siRNA comprises two or more gene-specific siRNAs (e.g. siRNA targeting ALOX5 or FLAP) as a pooled siRNA. In some embodiments, the siRNA comprises three or more gene-specific siRNAs (e.g. siRNA targeting ALOX5 or FLAP) as a pooled siRNA. In some embodiments, the siRNA comprises four or more gene-specific siRNAs (e.g. siRNA targeting ALOX5 or FLAP) as a pooled siRNA. In some embodiments, the siRNA comprises four gene-specific siRNAs (e.g. siRNA targeting ALOX5 or FLAP) as a pooled siRNA. Exemplary pooled siRNA is a SMARTpool siRNA (Dharmacon, Horizon Discovery). Different siRNAs formats, which in some embodiments are included as pooled siRNAs, are known in the art, including but not limited to siRNA with chemical modifications. Exemplary siRNA formats include ON-TARGETplus siRNA, Accell siRNA, siGENOME siRNA, or Lincode siRNA (Dharmacon, Horizon Discovery).
In some embodiments, the composition comprises a ALOX5 short-hairpin ribonucleic acid (shALOX5). For example, in various embodiments the composition is a nucleic acid vector encoding at least one of the shRNA designs targeting ALOX5. In various embodiments, the shRNA comprises a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the siRNA designs of ALOX5. In some embodiments, the composition comprises a FLAP short-hairpin ribonucleic acid (shFLAP). For example, in various embodiments the composition is a nucleic acid vector encoding at least one of the shRNA designs targeting FLAP. In various embodiments, the shRNA comprises a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the siRNA designs of FLAP.
In some embodiments, the composition comprises a guide RNA or a single-molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5. In some embodiments, the composition comprises a guide RNA or a single-molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the guide RNA or the single molecule guide RNA is pre-complexed with a DNA endonuclease. In some embodiments, the DNA endonuclease is a Cas9 or dCas9 endonuclease.
In some embodiments, the composition comprises a vector. In some embodiments, the vector is a viral vector wherein the viral vector encapsulates a nucleic acid encoding the shALOX5, a nucleic acid encoding the shFLAP, a nucleic acid encoding the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5, or a nucleic acid encoding the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the viral vector is an adeno-associated vector (AAV). In various embodiments the AAV is AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or variants thereof.
In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human subject has an increased expression level of ALOX5 compared to a subject not suffering from Huntington's disease. For example, expression of ALOX5 of a subject may be increased by at least 1.5 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. Expression of ALOX5 of a subject may be increased by at least 3 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. Expression of ALOX5 of a subject may be increased by at least 5 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. Expression of ALOX5 of a subject may be increased by at least 10 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease.
In some embodiments, the human subject has an increased expression level of FLAP compared to a subject not suffering from Huntington's disease. For example, expression of FLAP of a subject may be increased by at least 1.5 times greater than expression of FLAP of a subject not suffering from Huntington's disease. Expression of FLAP of a subject may be increased by at least 3 times greater than expression of FLAP of a subject not suffering from Huntington's disease. Expression of FLAP of a subject may be increased by at least 5 times greater than expression of FLAP of a subject not suffering from Huntington's disease. Expression of FLAP of a subject may be increased by at least 10 times greater than expression of FLAP of a subject not suffering from Huntington's disease.
In some embodiments, a population of cells can be contacted with a compound or agent which, for example, includes subjecting the cells to an appropriate culture media which comprises the indicated compound or agent. Where the cell population is in vivo, contacting the cell population includes administering the compound or agent in a pharmaceutical composition to a subject via an appropriate administration route such that the compound or agent contacts the cell population in vivo.
For in vivo methods, a therapeutically effective amount of a compound described herein can be administered to a subject. Methods of administering compounds to a subject are known in the art and easily available to one of skill in the art.
As described herein, the methods of treatment described herein refer generally to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. Methods described herein covers any treatment of a disease in a subject, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom, may or may not be diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
A therapeutically effective amount of an agent or composition disclosed herein, for example, is one that is effective for preventing, ameliorating, treating or delaying the onset of a disease or condition.
The pharmaceutical compositions of the inventions can be administered to any animal that can experience the beneficial effects of the agents of the invention. Such animals include humans and non-humans such as primates, pets and farm animals.
The present invention also comprises pharmaceutical compositions comprising the agents disclosed herein. Routes of administration and dosages of effective amounts of the pharmaceutical compositions comprising the agents are also disclosed. The agents of the present invention can be administered in combination with other pharmaceutical agents in a variety of protocols for effective treatment of disease.
Pharmaceutical compositions of the present invention are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. One may administer the viral vectors, siRNA, RNAi, shRNA or other inhibitors, or related compound in a local rather than systemic manner, for example, via injection of directly into the desired target site, often in a depot or sustained release formulation. Furthermore, one may administer the composition in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, the brain, and more specifically neuronal cells. The liposomes will be targeted to and taken up selectively by the desired tissue. Also included in a targeted drug delivery system is nanoparticle specific brain delivery of the viral vectors, siRNA, RNAi, shRNA or other inhibitors, or compound, alone or in combination with similar compounds. A summary of various delivery methods and techniques of siRNA administration in ongoing clinical trials is provided in Zuckerman and Davis 2015; Nature Rev. Drug Discovery, Vol. 14: 843-856, December 2015 the contents of which is hereby incorporated by reference in its entirety.
One of ordinary skill in the art will appreciate that a method of administering pharmaceutically effective amounts of the pharmaceutical compositions of the invention to a patient in need thereof, can be determined empirically, or by standards currently recognized in the medical arts. The agents can be administered to a patient as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents of the pharmaceutical compositions of the present invention will be decided within the scope of sound medical judgment by the attending physician. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. It is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
Dosages can also be administered in a patient-specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art.
ALOX5 or FLAP expression may be reduced using any known method in the art. For example, in various embodiments the composition is a viral vector encoding at least one of the siRNA designs targeting ALOX5 or FLAP. In various embodiments, the vector is a viral vector. In various embodiments, the viral vector is an AAV vector. In various embodiments, the viral vector is a vector that preferentially targets the brain or brain cells. In various embodiments the AAV is AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or variants thereof. In various embodiments, the subject is a mammal. In various embodiments, the mammal is a human. In various embodiments, the human has increased expression of ALOX5 or FLAP compared to a human not suffering from Huntington's disease.
In certain aspects, described herein is small interfering ribonucleic acid (siRNA) targeting the acyl-CoA synthetase long-chain family member 4 (ACSLA)-independent ferroptosis pathway. In some embodiments, the siRNA is an ALOX5 small interfering ribonucleic acid (siALOX5). In some embodiments, the siRNA is a FLAP small interfering ribonucleic acid (siFLAP). In certain aspects, described herein is a vector comprising a siRNA.
In certain aspects, described herein is a nucleic acid comprising a sequence encoding a short hairpin ribonucleic acid (shRNA) targeting the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway. In certain aspects, described herein is a vector comprising any of the nucleic acid described above. In certain aspects, described herein is a viral vector comprising the nucleic acid described above. In some embodiments, the viral vector is an AAV vector.
In some embodiment, targeted gene expression can be reduced by several genome editing techniques such as RNAi (RNA interference), zinc finger nucleases (ZFNs), a TALE-effector domain nuclease (TALLEN), CRISPR/Cas9 systems which are known in the art. In some embodiment, the CRISPR/Cas9 systems comprise a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA). In some embodiment, the gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX family. In some embodiment, the gRNA or sgRNA comprises a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP.
Inhibition of RNA encoding ALOX5 or FLAP can effectively modulate the expression of these proteins. Inhibitors can include siRNA; interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acids, which can be RNA, DNA, or an artificial nucleic acid.
Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding an EGFR fusion molecule can be synthesized, e.g., by conventional phosphodiester techniques. Antisense nucleotide sequences include, but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and the like.
siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA molecule. “Substantially identical” to a target sequence contained within the target mRNA refers to a nucleic acid sequence that differs from the target sequence by about 3% or less. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded “hairpin” area.
The siRNA can be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribo-nucleotides. One or both strands of the siRNA can also comprise a 3′ overhang. As used herein, a 3′ overhang refers to at least one unpaired nucleotide extending from the 3′-end of a duplexed RNA strand. For example, the siRNA can comprise at least one 3′ overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, or from 1 to about 5 nucleotides in length, or from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length. For example, each strand of the siRNA can comprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).
siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector. Methods for producing and testing dsRNA or siRNA molecules are known in the art.
RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs, which can function as antisense RNA. The ALOX5 or FLAP inhibitor can comprise ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded.
In some embodiments, the compositions is delivered through the blood brain barrier (BBB). Different methods for delivering compositions through the BBB, are known in the art, including but not limited to altering BBB integrity or osmotic disruption, conjugation to a biological carrier, use of shuttle compounds, use of stimuli responsive nanoparticles, hyperosmolar solutions, microbubbles and focused ultrasound (FUS), or receptor-mediated transcytosis (RMT).
In certain aspects, described herein is a composition for treating or preventing Huntington's disease, comprising an expression vector capable of targeting the ACSL4-independent ferroptosis pathway. In some embodiments, the composition reduces arachidonate 5-lipoxygenase (ALOX5) expression in a subject in need thereof. The expression of ALOX5 may be reduced by at least about 5% to about 95%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to corresponding expression of ALOX5 in a subject suffering from Huntington's disease or in the subject before administration of the composition. The expression of ALOX5 may be silenced relative to corresponding expression of ALOX5 in a subject suffering from Huntington's disease or in the subject before administration of the composition. In some embodiments, the expression of ALOX5 is reduced cells of the subject.
In some embodiments, the composition reduces 5-lipoxygenase-activating protein (FLAP) expression in a subject in need thereof. The expression of ALOX5 may be reduced by at least about 5% to about 95%, e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, relative to corresponding expression of FLAP in a subject suffering from Huntington's disease or in the subject before administration of the composition. The expression of FLAP may be silenced relative to corresponding expression of FLAP in a subject suffering from Huntington's disease or in the subject before administration of the composition. In some embodiments, the expression of FLAP is reduced cells of the subject.
In some embodiments, the composition comprises an ALOX5 small interfering ribonucleic acid (siALOX5). In some embodiments, the composition comprises a FLAP small interfering ribonucleic acid (siFLAP). In some embodiments, the composition comprises an ALOX5 short-hairpin ribonucleic acid (shALOX5). In some embodiments, the composition comprises a FLAP short-hairpin ribonucleic acid (shFLAP).
In some embodiments, the composition comprises a guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5. In some embodiments, the composition comprises a guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the guide RNA or the single molecule guide RNA is pre-complexed with a DNA endonuclease. In some embodiments, the DNA endonuclease is a Cas9 or dCas9 endonuclease.
In certain aspects, the composition further comprises a vector comprising the siRNA or shRNA or gRNA or sgRNAs described herein. In some embodiments, the composition further comprises a viral vector wherein the viral vector encapsulates a nucleic acid encoding the shALOX5, a nucleic acid encoding the shFLAP, a nucleic acid encoding the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding ALOX5, or a nucleic acid encoding the guide ribonucleic acid or a single molecule guide RNA comprising a spacer sequence that is complementary to a portion of a nucleic acid sequence encoding FLAP. In some embodiments, the viral vector is an AAV vector. In various embodiments the AAV is AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or variants thereof.
In certain aspects, described herein is small interfering ribonucleic acid (siRNA) targeting the acyl-CoA synthetase long-chain family member 4 (ACSLA)-independent ferroptosis pathway. In some embodiments, the siRNA is an ALOX5 small interfering ribonucleic acid (siALOX5). In some embodiments, the siRNA is a FLAP small interfering ribonucleic acid (siFLAP). In certain aspects, described herein is a vector comprising a SiRNA.
In certain aspects, described herein is a nucleic acid comprising a sequence encoding a short hairpin ribonucleic acid (shRNA) targeting the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway. In certain aspects, described herein is a vector comprising any of the nucleic acid described above. In certain aspects, described herein is a viral vector comprising the nucleic acid described above. In some embodiments, the viral vector is an AAV vector. In various embodiments the AAV is AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or variants thereof.
In some embodiments, the composition comprises Zileuton. Zileuton is FDA approved for treatment of asthma. The FDA prescribing label is available at FDA.gov and is hereby incorporated by reference in its entirety.
In some embodiments, the composition comprises (±)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea or pharmaceutically acceptable salts thereof. In some embodiments, the composition comprises (±)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea. In some embodiments, the composition comprises
In some embodiments, the composition consists of (±)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea or pharmaceutically acceptable salts thereof. In some embodiments, the composition consists of (±)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea. In some embodiments, the composition consists of
In some embodiments, the composition consists essentially of (±)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea or pharmaceutically acceptable salts thereof. In some embodiments, the composition consists essentially of (±)-1 (1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea. In some embodiments, the composition consists essentially of
In some embodiments, the composition comprises a tablet comprising Zileuton. In some embodiments, the tablet is as an extended release tablet. In some embodiments, the composition comprises a tablet comprising 600 mg of Zileuton. In some embodiments, the composition comprises a tablet comprising 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg or more of Zileuton. In some embodiments, the composition is administered at a dose of 2400 mg daily. In some embodiments, the composition is administered at a dose of 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, or 3000 mg or more daily. In some embodiments, the composition is administered as two 600 mg tablets twice daily (or equivalents thereof e.g four 300 mg tablets twice daily). In some embodiments, the composition is administered within one hour after morning and evening meals.
In some embodiments, the composition comprises
or pharmaceutically acceptable salts thereof. In some embodiments, the composition comprises
In some embodiments, the composition consists of
or pharmaceutically acceptable salts thereof. In some embodiments, the composition consists of
In some embodiments, the composition consists essentially of
or pharmaceutically acceptable salts thereof. In some embodiments, the composition consists essentially of
In some embodiments, the composition comprises docebenone (AA 861). is a potent, selective and orally active 5-LO (5-lipoxygenase) inhibitor. In some embodiments, the composition comprises
In some embodiments, the composition comprises boswellic acids. 11-keto-boswellic acids inhibit ALOX5. In some embodiments, the composition comprises atreleuton (ABT-761 or VIA-2291). Atreleuton is a selective, reversible, and orally bioavailable ALOX5 inhibitor. In some embodiments, the composition comprises
In some embodiments, the composition comprises setileuton (1,3,4-oxadiazole MK-0633). Setileuton (1,3,4-oxadiazole MK-0633), a potent and selective ALOX5 inhibitor. In some embodiments, the composition comprises
In some embodiments, the composition comprises PF-4191834 or CJ-13610. In some embodiments, the composition comprises
In some embodiments, the composition comprises flavocoxid. Flavocoxid is a mixture of purified bioflavonoids that inhibits COX-1, COX-2, and ALOX5 in vitro and in animal models. Pharmaceutical compositions of the present invention are administered to a subject in a manner known in the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. One may administer the viral vectors, siRNA, RNAi, shRNA or other inhibitors, or related compound in a local rather than systemic manner, for example, via injection of directly into the desired target site, often in a depot or sustained release formulation. Furthermore, one may administer the composition in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, the brain, and more specifically neuronal cells. The liposomes will be targeted to and taken up selectively by the desired tissue. Also included in a targeted drug delivery system is nanoparticle specific brain delivery of the viral vectors, siRNA, RNAi, shRNA or other inhibitors, or compound, alone or in combination with similar compounds. A summary of various delivery methods and techniques of siRNA administration in ongoing clinical trials is provided in Zuckerman and Davis 2015; Nature Rev. Drug Discovery, Vol. 14: 843-856, December 2015 the contents of which is hereby incorporated by reference in its entirety.
One of ordinary skill in the art will appreciate that a method of administering pharmaceutically effective amounts of the pharmaceutical compositions of the invention to a patient in need thereof, can be determined empirically, or by standards currently recognized in the medical arts. The agents can be administered to a patient as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents of the pharmaceutical compositions of the present invention will be decided within the scope of sound medical judgment by the attending physician. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. It is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
Dosages can also be administered in a patient-specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art.
In various embodiments, the present application discloses compositions for regulating the ferroptosis through the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway. In various embodiments, the present application discloses a composition that reduces ALOX5 expression or function. In various embodiments, the present application discloses a composition that inhibits FLAP expression or function. In various embodiments, the present application discloses a composition comprising ALOX5 siRNA. In various embodiments, the present application discloses a composition comprising FLAP siRNA. For example, in various embodiments the composition is a vector comprising at least one of the small interfering ribonucleic acid (siRNA) designs targeting ALOX5 or FLAP. In various embodiments, the siRNA is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the siRNA designs of ALOX5 or FLAP. In various embodiments, the vector is a viral vector. In various embodiments, the viral vector is an AAV vector. In various embodiments, the viral vector is a vector that preferentially targets the brain or brain cells. In various embodiments the AAV is AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or variants thereof.
In various embodiments, the present application discloses compositions for regulating the ferroptosis through the acyl-CoA synthetase long-chain family member 4 (ACSL4)-independent ferroptosis pathway. In various embodiments, the present application discloses a composition that reduces ALOX5 expression or function. In various embodiments, the present application discloses a composition that inhibits FLAP expression or function. In various embodiments, the present application discloses a composition comprising ALOX5 siRNA. In various embodiments, the present application discloses a composition comprising FLAP siRNA. For example, in various embodiments the composition is a vector comprising at least one of the small interfering ribonucleic acid (siRNA) designs targeting ALOX5 or FLAP. In various embodiments, the siRNA is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the siRNA designs targeting ALOX5 or FLAP. In various embodiments, the vector is a viral vector. In various embodiments, the viral vector is an AAV vector. In various embodiments, the viral vector is a vector that preferentially targets the brain or brain cells. In various embodiments the AAV is AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or variants thereof.
In certain aspects, the invention provides a method of diagnosing Huntington's disease in a subject comprising, determining the presence of mutant huntingtin (mHTT) in the subject. In certain aspects, the invention provides a method of diagnosing Huntington's disease in a subject comprising (i) determining the level of arachidonate 5-lipoxygenase (ALOX5) in the subject; and (ii) diagnosing the subject with Huntington's disease if the level of ALOX5 expression in the subject is increased as compared to a subject not suffering from Huntington's disease. For example, expression of ALOX5 of a subject may be increased by at least 1.5 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. Expression of ALOX5 of a subject may be increased by at least 3 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. Expression of ALOX5 of a subject may be increased by at least 5 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. Expression of ALOX5 of a subject may be increased by at least 10 times greater than expression of ALOX5 of a subject not suffering from Huntington's disease. In some embodiments, the method further comprises determining the level of FLAP in the subject. In some embodiments, the method further comprises determining the whether the subject expresses mHTT. In certain aspects, the invention provides a method of diagnosing Huntington's disease in a subject comprising (i) determining determining the level of 5-lipoxygenase-activating protein (FLAP) in the subject; and (ii) diagnosing the subject with Huntington's disease if the level of FLAP expression in the subject is increased as compared to a subject not suffering from Huntington's disease. For example, expression of FLAP of a subject may be increased by at least 1.5 times greater than expression of FLAP of a subject not suffering from Huntington's disease. Expression of FLAP of a subject may be increased by at least 3 times greater than expression of FLAP of a subject not suffering from Huntington's disease. Expression of FLAP of a subject may be increased by at least 5 times greater than expression of FLAP of a subject not suffering from Huntington's disease. Expression of FLAP of a subject may be increased by at least 10 times greater than expression of FLAP of a subject not suffering from Huntington's disease. In some embodiments, the method further comprises determining the level of ALOX5 in the subject. In some embodiments, the method further comprises determining whether the subject expresses mHTT.
In some embodiments the level of ALOX5, FLAP, and/or mHTT is determined from a sample from the subject. In some embodiments, Huntington disease is diagnosed in the subject by determining ALOX5 mRNA levels, ALOX5 protein levels, or both ALOX5 mRNA levels and ALOX5 protein levels in a sample from the subject. In some embodiments, Huntington disease is diagnosed in the subject by determining FLAP mRNA levels, FLAP protein levels, or both FLAP mRNA levels and FLAP protein levels in a sample from the subject. In some embodiments, the subject is diagnosed with Huntington's disease if the level of both FLAP and ALOX5 expression in the subject is increased as compared to a subject not suffering from Huntington's disease. In some embodiments, the subject is diagnosed with Huntington's disease if the level of FLAP expression in the subject is increased as compared to a subject not suffering from Huntington's disease and the subject expresses mHTT. In some embodiments, the subject is diagnosed with Huntington's disease if the level of ALOX5 expression in the subject is increased as compared to a subject not suffering from Huntington's disease and the subject expresses mHTT. In some embodiments, the subject is diagnosed with Huntington's disease if the level of both ALOX5 and FLAP expression in the subject is increased as compared to a subject not suffering from Huntington's disease and the subject expresses mHTT. In some embodiments, the invention comprises detecting in a biological sample whether there is an increase in an mRNA encoding ALOX5 or FLAP. Methods for detecting and quantifying ALOX5 or FLAP molecules in biological samples are known the art. For example, protocols for detecting and measuring a ALOX5 or FLAP protein molecule using either polyclonal or monoclonal antibodies specific for the polypeptide are well established. Non-limiting examples include Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). Methods for detecting and quantifying ALOX5 or FLAP mRNA in biological samples are known the art. In some embodiments, the level of mRNA is determined using RT-qPCR or RNA-seq.
In one aspect, the invention provides a device for determining whether a sample from a subject contains ALOX5 or FLAP protein, or a combination thereof, the device comprising at least one antibody that specifically binds to ALOX5 or FLAP protein, or a fragment thereof. In another aspect, the invention provides a device for determining whether a sample from a subject contains ALOX5 or FLAP nucleic acid, or a combination thereof, the device comprising at least one primer, primer pair, or nucleic acid probe, that specifically binds to ALOX5 or FLAP nucleic acid, or a fragment thereof.
In one embodiment, a biological sample comprises, a blood sample, serum, cells (including whole cells, cell fractions, cell extracts, and cultured cells or cell lines), tissues (including tissues obtained by biopsy), body fluids (e.g., urine, sputum, amniotic fluid, synovial fluid), or from media (from cultured cells or cell lines). In some embodiments, the sample is blood or blood serum or CSF. In some embodiments, the same is cells of the subject. In some embodiments, the sample is nervous system tissue of the subject. In some embodiments, the sample comprises brain cells of the subject. The methods of detecting or quantifying a ALOX5 or FLAP molecule include, but are not limited to, amplification-based assays with (signal amplification) hybridization based assays and combination amplification-hybridization assays. For detecting and quantifying a ALOX5 or FLAP molecule, an exemplary method is an immunoassay that utilizes an antibody or other binding agents that specifically bind to an ALOX5 or FLAP protein or epitope of such, for example, Western blot or ELISA assays. Methods for detecting and quantifying ALOX5 or FLAP mRNA in biological samples are known the art. In some embodiments, the level of mRNA is determined using RT-qPCR or RNA-seq.
In some embodiments, the compositions is delivered through the blood brain barrier (BBB). Different methods delivering compositions through the BBB, are known in the art, including but not limited to altering BBB integrity or osmotic disruption, conjugation to a biological carrier, use of shuttle compounds, use of stimuli responsive nanoparticles, hyperosmolar solutions, microbubbles and focused ultrasound (FUS), or receptor-mediated transcytosis (RMT).
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield similar results.
Although it is well-established that Huntington's disease (HD) is mainly caused by polyglutamine expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT showed that it is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSLA-dependent ferroptosis has no obvious effect on the life span of Huntington's disease mouse. Moreover, by using RNAi-mediated screening, ALOX5 was identified as a major factor required for the ACSLA-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.
Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by progressive cognitive, behavioral, motor dysfunctions and short life-spans (Bates et al. 2015). HD is caused by a CAG trinucleotide repeat expansion in Exon 1 of the HTT gene, which results in an expanded polyglutamine (polyQ) tract in the encoded huntingtin protein, referred to as mutant huntingtin (mHTT). The mHTT exhibits toxic gain of functions causing neuronal dysfunction and cell death (MacDonald et al. 1993; Bates 2003). The proteolytic cleavage of mHTT is a key event in the molecular pathogenesis of HD and it is believed that the N-terminal fragment containing the expanded polyglutamine of mHTT plays an important role in the pathogenesis of HD (Martindale et al. 1998; Lunkes et al. 2002; Graham et al. 2006). Although the genetic cause of HD is well established, the cellular and molecular mechanisms involved in mHTT-mediated early neuronal dysfunction and late neurodegeneration are not fully understood.
Redox signaling is essential for normal brain function, being involved in memory consolidation, neuronal differentiation, and plasticity. Ferroptosis is a regulated form of non-apoptotic cell death driven by excess accumulation of lipid peroxidates critically regulated by the redox signaling (Stockwell et al. 2020). Accumulating evidence indicates that ferroptosis may be involved in both animal models and human patients with Huntington's disease. For example, in transgenic HD mouse models and patients, most of neuronal death did not exhibit the classic apoptotic features (Turmaine et al. 2000; Hickey and Chesselet 2003). Moreover, higher levels of lipid peroxidation are observed as a principal characteristic in HD patients (Klepac et al. 2007). It was reported that increased levels of lipid peroxidation were detected in corticostriatal brain slices (Skouta et al. 2014) and were colocalized with mHTT inclusions in the striatal neurons (Lee et al. 2011). Inhibition of lipid peroxidation with ferrostatin-1(Ferr-1) significantly rescued the cell death in cellular models of Huntington's disease (HD) (Skouta et al. 2014). On the other hand, lower GSH levels are another characteristics in HD patients (Klepac et al. 2007). Indeed, decreased GSH and GSH-S-transferase were detected in the striatum, cortex and hippocampus in 3-nitropropionic acid (3-NP)-induced HD mouse (Kumar et al. 2010). mHTT can directly interact with mitochondrial proteins, such as translocase of the inner membrane 23 (TIM23), disrupting mitochondrial proteostasis and favoring ROS production and HD progression (Yablonska et al. 2019). Thus, it is very important to examine whether mHTT is directly involved in regulating ferroptosis and more importantly, the molecular factors that mediate mHTT-dependent ferroptosis need to be delineated.
Ferroptosis, an iron-dependent form of non-apoptotic cell death driven by lipid-based reactive oxygen species (ROS), is tightly linked with human diseases including neurodegenerative diseases (Stockwell, 2020). Lipid peroxides can be eliminated by glutathione peroxidase 4 (GPX4) and its co-factor glutathione (GSH) (Stockwell et al., 2017). Thus, ferroptosis is commonly induced by pharmacological agents that disrupt this lipid repair system, that directly or indirectly lead to GPX4 inhibition (Stockwell et al., 2017). Notably, by using genome-wide haploid and CRISPR-Cas9-based screening, acyl-CoA synthetase long-chain family member 4 (ACSL4) was identified as an essential factor for ferroptosis induced by GPX4 inhibitors (Doll et al. 2017; Kagan et al. 2017). The critical role of ACSL4 in ferroptosis relies on its ability to incorporate arachidonic acid and adrenic acid into phosphatidylethanolamines and thereby provides the main substrates for peroxidation (Kagan et al. 2017). In addition to GPX4-mediated neutralization of lipid peroxidation, the levels of cellular lipid peroxides can be induced enzymatically by the lipoxygenases. Indeed, recent studies showed that the ferroptotic response induced by the ALOX12 lipoxygenase plays an important role in catalyzing lipid peroxidation and p53-mediated effect on Myc-induced lymphomagenesis (Chu et al., 2019). Together, these studies indicate that two types of ferroptosis are critical for oxidative stress responses: ACSL4-dependent ferroptosis is induced by these common ferroptosis inducers mainly including GPX4 inhibitors whereas ACSL4-independent ferroptosis is induced by high levels of reactive oxygen species (ROS).
Here, expression of the mHTT fragment with the expanded 94 glutamine residues (HTTQ94) showed that it directly promotes ferroptosis in both neuronal cells and neuroblastoma cells upon erastin treatment. As expected, inhibition of ACSL4 in those cells completely abrogated the ferroptotic response induced by HTTQ94; surprisingly, however, inactivation of the acsl4 gene failed to show any significant effect in the Huntington's disease transgenic mouse model (HD-N171-82Q). Moreover, ALOX5 was identified as a major mediator for HTTQ94-driven ferroptosis upon ROS-induced stress. Interestingly, neither ACSL4 nor GPX4 is required for ALOX5-dependent ferroptosis induced by HTTQ94. Moreover, knockout of the Alox5 gene abrogated ferroptosis in the striatal neurons from the HD mice and significantly improved the pathological phenotypes of Huntington's disease mice. Thus, the data described herein reveals a novel ferroptosis pathway critically involved in the pathological mechanism of Huntington's disease.
To elucidate the potential role of ferroptosis in Huntington's disease, whether mHTT is directly involved in regulating ferroptotic responses was first examined. To this end, the mouse hippocampal neuronal cell line HT-22 was used to establish HTTQ94 tet-on inducible cell lines, in which expression of the N-terminal fragment mutant huntingtin protein containing the expanded 94 glutamine residues (HTTQ94) can be induced by doxycycline. As shown in
Recent studies indicate that acyl-CoA synthetase long-chain family member 4 (ACSL4) is essential for the ferroptotic responses induced by either erastin or GPX4 inhibitors (Doll et al. 2017; Kagan et al. 2017; Stockwell et al. 2017). To dissect the molecular mechanism of mHTT mediated ferroptosis, the function of the ACSLA inhibitors in HTTQ94-mediated ferroptosis was first examined. Both the ACSL4 inhibitors, rosiglitazone (ROSI) and troglitazone (TRO), prevented HTTQ94 mediated ferroptosis in both HT-22 and SK-N-BE(2)C cells (
By establishing Acsl4 knockout mice, the GPX4-dependent ferroptosis can also be inactivated upon loss of ACSL4 expression in vivo (Chu et al. 2019). To further elucidate the role of mHTT mediated ferroptosis in pathogenesis of Huntington's disease, whether inactivation of the GPX4 dependent ferroptosis has any effect on the phenotypes observed in Huntington's disease mouse models was examined. To this end, a well-established HD transgenic mouse model (HD-N171-82Q), in which a HTTQ82 protein with the N-terminal fragment (171 amino acids) and an 82 residue glutamine repeat was constitutively expressed (Schilling et al. 1999) was used. These mice developed behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait and died prematurely within ˜120 days after birth (Schilling et al. 1999). After crossed these transgenic HD mice with Acsl4 knockout mice, the HD/Acsl4-null mice was successfully generated. As expected, the acsl4 protein was undetectable in the brain of the HD/Acsl4-null mice (
The previous study showed that the ferroptotic response can also be induced upon ROS stress, independent of the ACSL4 status (Chu et al. 2019). Since numerous studies indicate the importance of ROS stress in Huntington's disease pathogenesis (Paul and Snyder 2019), whether HTTQ94 promotes ROS-induced ferroptosis in the established HT-22 HTTQ94 inducible cell line upon ROS-induced stress, generated by tert-butyl hydroperoxide (TBH) was first examined. As shown in
Next, whether this type of ferroptosis is dependent on ACSL4 by using the established ACSL4 knockout cell lines was examined. Indeed, the ferroptosis response induced by HTTQ94 remained intact in ACSL4-null cells under the same conditions (
Finally, in addition to GPX4, several important cellular factors have been identified critically involved in regulating ferroptotic responses through completely different mechanisms such as FSP1, GCH1 and DHODH (Doll et al. 2019; Bersuker et al. 2019; Kraft et al. 2020; Mao et al. 2021). To examine whether these ferroptosis regulators play a role in HTTQ94-mediated ferroptosis upon ROS stress, RNAi-mediated depletion in the tet-on HTTQ94-inducible SK-N-BE(2)C cells (
Notably, the recent studies showed that ferroptosis can be induced by activation of the lipoxygenases independent of these known factors (Chu et al. 2019). The mammalian lipoxygenase family consists of six isoforms (ALOXE3, ALOX5, ALOX12, ALOX12B, ALOX15, and ALOX15B) with differing substrate specificities (Mashima and Okuyama 2015). To examine whether any of these lipoxygenases is required for mHTT-mediated ferroptosis upon ROS stress, an RNAi-mediated loss-of-function screen was performed to test whether depletion of individual isoforms affects HTTQ94 dependent ferroptosis. To this end, the tet-on HTTQ94-inducible SK-N-BE(2)C cells were transfected with siRNAs (Dharmacon SMART-pools) specific for each of the six lipoxygenases and treated with TBH to induce ferroptosis. Significantly, mHTTQ94-mediated ferroptosis upon ROS stress was markedly blocked by RNAi-mediated depletion of ALOX5, but not by the depletion of the other five lipoxygenases (
To further validate the role of ALOX5 in HTTQ94-mediated ferroptosis, ALOX5 knockout cells by CRISPR/cas9 technology was generated by using the HTTQ94 tet-on SK—N-BE(2)C cells (
Glutamate, the most abundant endogenous excitatory neurotransmitter in brain, plays a crucial role in neuronal tissue damage. Recently, several studies showed that ferroptosis is regulated by glutamate (Liu et al. 2015; Maher et al. 2020; Xie et al. 2022). The HTTQ94 inducible HT-22 cell line was treated with glutamate to investigate whether HTTQ94 also promotes glutamate induced ferroptosis. Indeed, high levels of cell death were induced upon HTTQ94 expression in the presence of glutamate (
To dissect the mechanism by which HTTQ94 promotes ALOX5-dependent ferroptosis, whether the protein levels of ALOX5 is regulated by HTTQ94 expression was tested. As shown in
Next, the interaction between HTTQ94 (or HTTQ19) and FLAP was examined in human cells. To this end, cells were transfected with a HTTQ94 (or HTTQ19) expression vector in the presence or absence of a vector encoding Flag-FLAP in 293 cells. As shown in
To examine the role of HTTQ94-mediated, ALOX5-dependent ferroptosis in contributing to the pathophysiological phenotypes in the HD mice, these HD mice (HD-N171-82Q) were crossed with Alox5-null mice to generate compound HD/Alox5null mutant mice. As shown in
To evaluate the role of ferroptosis in contributing to the pathological phenotypes of these mice, whether mHTT-mediated ferroptosis is indeed elevated in the HD mice and whether loss of ALOX5 is sufficient to abrogate the ferroptotic response in vivo were examined. Ferroptosis is a new type of programed cell death driven by the iron-dependent accumulation of lipid ROS. To further examine the levels of ferroptosis in the HD mice, a reliable way to specifically recognize ferroptotic cells in tissue sections to characterize the extent of ferroptosis in animal models had to be established. Notably, by screening ˜4,750 of the resulting monoclonal antibodies generated for their ability to selectively detect cells undergoing ferroptosis, one antibody, 3F3 ferroptotic membrane antibody (3F3-FMA) identified as the human transferrin receptor 1 protein (TfR1), is effective as a specific ferroptosis-staining reagent (Feng et al. 2020).
To validate whether this ferroptosis marker is effective at staining ferroptotic cells in the HD mice, whether the anti-TfR1 antibody staining is able to specifically recognize ferroptotic cells induced by HTTQ94 expression was first examined in mouse neuronal cells. To this end, the tet-on-HTTQ94 mouse hippocampal HT-22 neuronal inducible cell line was used. As shown in
Cell death induced by mHTT toxicity is a pathological hallmark in HD, characterized by significant neuronal loss in the striatum and cerebral cortex, followed by widespread neuronal loss in other brain regions (Thu et al. 2010; Nana et al. 2014; Bates et al. 2015). Previous studies have indicated that apoptosis is apparently not the major factor for the neuronal cell death in HD (Turmaine et al. 2000; Hickey and Chesselet 2003). Ferroptosis is a newly identified form of cell death that is morphologically, biochemically, and genetically distinct from other known forms of cell death (Dixon et al. 2012; Stockwell et al. 2017). Indeed, that expression of the mHTT fragment with the expanded 94 glutamine residues (HTTQ94) but not the wild type counterpart (HTTQ19) sensitizes neuronal cells to ferroptosis was found. Although numerous studies implicate ACSLA as a central factor in modulating ferroptotic responses induced by GPX4 inhibition, inactivation of the ACSL4-dependent ferroptosis did not have significant effect on pathophysiological phenotypes and the life span of the HD mice. Notably, HTTQ94 can also induce ferroptosis upon ROS-induced stress through a distinct regulatory pathway. Further, HTTQ94 mediated ferroptosis upon ROS stress is mediated by ALOX5. Moreover, HTTQ94 activates ALOX5 function through stabilizing its cofactor FLAP.
Signs and symptoms of Huntington's disease typically begin ages 30 to 50 and progress over the next 10 to 20 years (Bates et al. 2015), suggesting that besides mHTT toxic function, aging also plays an important role in neuronal death. The production of ROS is progressively increased in aging which is one of the key factors in cellular damage (Floyd and Hensley 2002; Tower 2015). Although the precise mechanism of mHTT-mediated ferroptosis in vivo needs further elucidation, based on the data described herein, it is very likely that mHTT-mediated ferroptotic responses are tightly regulated by the levels of ROS production in HD patients. Moreover, glutamate, the most common endogenous excitatory neurotransmitter in brain, plays a crucial role in neuronal tissue damage. Under normal physiological condition, glutamate regulates memory, learning, cognitive, emotional, endocrine and other visceral functions (Liu et al. 2015; Maher et al. 2020; Xie et al. 2022). Interestingly, the data described herein indicate that glutamate-induced ferroptosis is significantly enhanced by HTTQ94 expression. More importantly, the results show that ALOX5 is essential for these effects. Numerous studies indicate that glutamate-mediated cell death is involved in the pathogenesis of almost all neurological diseases, including Huntington's diseases (Coyle and Puttfarcken 1993; Caudle and Zhang 2009; Lewerenz and Maher 2015; McGrath et al. 2022). These data reveal a potential physiological pathway in modulating mHTT-induced ferroptosis during the pathological process of Huntington's diseases. Ferroptosis driven by lethal amounts of lipid peroxide is critical for ROS stress responses (Yang and Stockwell 2016). Lipid peroxidation has been observed in the brain of the HD mice (Lee et al. 2011; Skouta et al. 2014), indicating the involvement of ferroptosis in the progression of HD. The data described herein further showed that loss of ALOX5 expression abrogates ferroptosis in the striatal neurons of the HD mice. More importantly, the HD/Alox5″ull mutant mice survived significantly longer than HD mice with much improved pathological phenotypes. Phenotypes of the HD/Alox5″ull mutant mice can be further characterized and findings in the HD mouse model expressing full-length mHTT and other HD mouse models can be further validated using this model. Nevertheless, the findings described herein indicate that ALOX5-dependent ferroptosis pathway is crucial of mHTT-induced pathophysiological phenotypes of the HD mice and suggest that inhibition of the ALOX5 activity may be beneficial for treatment of Huntington's disease.
Although the role of the ACSL4-mediated ferroptosis in contributing to several types of neurodegenerative diseases has been well recognized, the data described herein implicates that a new ferroptosis pathway controlled by ALOX5 is critical for mHTT-mediated effects in HD mice. There are several additional mouse models of HD including the BAC transgenic or Knockin mice that express full length HTT. HdhQ111 mice accurately express 111-glutamine mutant huntingtin and exhibit early dominant abnormalities selective for medium spiny striatal neurons, including nuclear retention of full-length mutant huntingtin (Wheeler et al., 2000). A bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) was developed expressing full-length-mHtt with 97 glutamine repeats under the control of endogenous htt regulatory machinery on the BAC (Gray et al., 2008). Both HD mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuron degeneration. Whether loss of ALOX5 expression is able to effectively ameliorate pathological phenotypes and significantly extend the life spans of HD mice can be further studied. Moreover, mHTT-mediated ferroptosis is effectively blocked by Zileuton, a specific inhibitor of ALOX5. Interestingly, Zileuton is a FDA-approved drug that has been used for the treatment of asthma (Liu et al., 2015). The effect of this compound in the pathological phenotypes and the life spans of HD mice can be tested for repurposing this drug to therapeutic application in Huntington's disease.
The SK-N-BE(2)C neuroblastoma, H1299 and HEK293 cell lines were obtained from American Type Culture Collection (ATCC). The HT-22 Mouse Hippocampal Neuronal Cell Line was obtained from Sigma-Aldrich in 2018. All cells have been proven to be negative for mycoplasma contamination. No cell lines used in this work were listed in the ICLAC database. All cells were cultured in a 37° C. incubator with 5% CO2. All media were supplemented in DMEM with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Gibco). Stable cell lines derived from these cell lines and experimental treatments are described in method details.
pTreTight-HTTQ94-GFP was a gift from Nico Dantuma (Addgene plasmid #23966; http://n2t.net/addgene:23966; RRID: Addgene_23966). HTTQ94-GFP was subcloned into pcDNA3.1/v5-His-Topo vector (Invitrogen). HTTQ94 sequence was amplified from pTreTight-HTTQ94-GFP. HTTQ19 (nHTT) sequence was amplified from the DNA isolated from Human cells. Both the HTTQ94 and HTTQ19 sequences were subcloned into pDONR221-vector and then were subcloned into pInducer-vector (Gateway LR kit, Thermo) to generate pInducer-HTTQ19 and pInducer-HTTQ94 respectively. V5-ALOX5 was previously described (Chu et al., 2019). pDONR221-FLAP was obtained from Harvard Medical School PlasmID (HsCD00043730). To generate HA-FLAP and Flag-FLAP, full length FLAP was amplified using forward primer with HA sequence and Flag sequence respectively, and then was subcloned into pcDNA3.1/v5-His-Topo vector (Invitrogen). cDNA of AIFM2 (FSP1), GCH1 and DHODH was amplified using forward primer with Flag sequence and cloned into pcDNA3.1/v5-His-Topo vector (Invitrogen).
To generate the HTTQ94 and HTTQ19 fragments Tet-on stable cell lines, pInducer-HTTQ94 and pInducer-HTTQ19 were transfected into H1299 cells. To generate the HTTQ94 Tet-on stable neuronal cell lines, pInducer-HTTQ94 was transfected into HT-22 and SK-N-BE(2)C cells. The transfected cells were selected and maintenanced with 500 μg/mL G418 (Sigma) in DMEM medium containing 10% FBS. Single clones were selected and screened by western blot. ACSL4 and ALOX5 CRISPR-cas9-knockout cells were generated by transfecting ACSL4 and ALOX5 double nickase plasmid (ACSL4, sc-401649-NIC; ALOX5, sc-401239-NIC; Santa Cruz) into the HTTQ94 Tet-on SK-N-BE(2)C cells. ACSL4/GPX4 double-knockout cells were generated by transfecting ACSL4 and GPX4 double nickase plasmid (GPX4, sc-401558-NIC; Santa Cruz) simutaneously into the HTTQ94 Tet-on SK-N-BE(2)C cells. Forty-eight hours later, CRISPR efficiency was determined by Western blot analysis, and then pool cells were seeded in 10-cm dish to grow clones at a density of 100-200 cells/dish. One or two weeks later, moloclones were picked and seeded into 12-well plates, followed by identification by Western blot analysis.
Protein extracts were analyzed by Western Blotting according to standard protocols using primary antibodies specific for ALOX5 (3289S; Cell signaling; 1:500 dilution), ACSL4 antibody (A5) (sc-271800; Santa Cruz; 1:1000 dilution), HA (11867423001; Sigma; 1:1000 dilution), Flag (F-3040;Sigma; 1:1000 dilution), FLAP (ab85227; Abcam; 1:1000 dilution), β-actin (Ab8227; Abcam; 1:2000 dilution) and vinculin (V9264; Sigma-Aldrich; 1:5000 dilution). HRP-conjugated anti-mouse (1031-05; SouthernBiotech) and anti-rabbit secondary antibody (4050-05; SouthernBiotech) and anti-rat secondary antibody (3051-05; Southern Biotech) were used.
Cells were plated at 20˜30% density one day prior to siRNAs transfection. Knockdown of ALOX family proteins and FLAP was performed by transfection of HTTQ94 Tet-on SK-N-BE(2)C cells with 60 μM siRNA duplex oligoset (ON-TARGET plus SMARTpool: ALOXE3: L-009022-00-0005; ALOX12B: L-009025-00-0005; ALOX12: L-004558-00-0005; ALOX5: L-004530-00-0005; ALOX15B: L-009026-00-0005; ALOX15: L-003808-00-0005; FLAP: L-010166-01-0005; Horizon Discovery) with Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. HTTQ94 Tet-on HT-22 cells were transfected with 60 μM mouse ALOX5 siRNA duplex oligo set (ON-TARGET plus SMARTpool, L-065695-01-0005, Horizon Discovery). HTTQ94 Tet-on SK-N-BE(2)C cells were transfected with 40 μM AIFM2/FSP1, GCH1 and DHODH siRNAs pool (Invitrogen, Shanghai, China). Cells were collected at 48˜72 h after transfection and subjected to functional assays. The siRNA target sequences were follows:
RNA Extraction and qRT-PCR.
Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Cat #15596018) according to the manufacturer's protocol. cDNA was generated using SuperScript IV VILO Master Mix (Thermo Fisher Scientific, Cat #11756500). Quantitative PCR was done using a 7500 Fast Real-Time PCR System (Applied Biosystems) with standard protocol. Reactions were done in triplicate. The following primers were used: human ALOX family and mouse CHAC1 described previously (Chu et al., 2019); human FLAP forward 5′-CTTGCCTTTGAGCGGGTCTA-3′, reverse 5′-CATCAGTCCAGCAAACGCAG-3′; mouse ALOX5 forward 5′-ATCGAGTTCCCATGTTACCGC-3′, reverse 5′-AATTTGGTCATCTCGGGCCA-3′; human AIFM2/FSP1 forward 5′-AGACAGGGTTCGCCAAAAAGA-3′, reverse 5′-CAGGTCTATCCCCACTACTAGC-3′; human GCH1 forward 5′-ACGAGCTGAACCTCCCTAAC-3′, reverse 5′-GAACCAAGTGATGCTCACACA-3′; human DHODH forward 5′-GTTCTGGGCCATAAATTCCGA-3′, reverse 5′-TCTGGGTCTAGGGTTTCCTTC-3′.
All drugs (except for drugs below) were ordered from Sigma-Aldrich. Ferrostatin-1 was from Xcess Biosciences; MK886 were from MedChemExpress (MCE); For ROS generation, tert-butyl hydroxide solution (TBH) was used at different doses depending on the experiment; see respective figure legends. Erastin (ferroptosis inducer), Rosiglitazone (ACSL4 inhibitor), Troglitazone (ACSLA inhibitor), Zileuton (ALOX5 inhibitor), MK886 (FLAP inhibitor), Ferrostatin-1 (ferroptosis inhibitor), DFO (ferroptosis inhibitor), Liproxstatin-1 (ferroptosis inhibitor), 3-methylademine (autophagy inhibitor), Z-VADFMK (apoptosis inhibitor) and necrostatin-1(necroptosis inhibitor) were used at different doses depending on the experiment; see related figure legends.
Cells were treated with ferroptosis inducer TBH or erastin or glutamate combined with other drugs or conditions. At indicated time points, cells were trypsinized and stained with trypan blue followed by counting with a hemocytometer using the cell number counter (Life technologies countess II). Living cells and dead cells were all counted, and cells stained blue were considered as dead cells. Quantification of cell death was further confirmed using ToxiLight Non-destructive Cytotoxicity BioAssay Kit (LT07-117; Lonza). Data were collected using GloMax® Explorer Multimode Microplate Reader (Promega).
Cells were incubated with DMEM containing 5 μM of BODIPY™ 581/591 C11 (D3861; Thermo Fisher Scientific) at the concentration of 5 μM for 25-30 min at 37° C. in serum-free medium. Cells were then harvested and washed twice with PBS followed by re-suspending in 500 μl of PBS. Lipid ROS levels were analyzed using a Becton Dickinson FACS Calibur machine through the FL1 channel, and the data were analyzed using CellQuest software. 10000 cells were collected and analyzed for each sample.
The Alox5 knockout mice and Huntington transgenic mouse (HD-N171-82Q) were purchased from the Jackson Laboratory (stock number 004155 and 003627, respectively). The Acs14 knockout mice were described previously (Chu et al., 2019) . . . . All experimental protocols using mice were approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
For cell line: HT-22 HTTQ94 inducible cells were fixed with 2% paraformaldehyde at RT for 20 min, and then incubated with blocking buffer (10% donkey serum, 0.3% Triton X-100 in PBS), followed by incubation with TfR1 antibody (1: 250, Thermo Fisher Scientific, Cat #13-6800) for 1 h, and then incubation with Alexa 568 conjugated secondary antibody (1:250, Thermo Fisher Scientific, Cat #A11004) for additional 1 h.
For mouse brain slides: brains paraffin slides were dewaxed by xylene, and antigens were retrieved with PH6.0 citrate buffer using pressure cooker. Slides were blocked with 5% horse serum for 30 min, and then incubated with primary antibody (TfR1: 1:1000, Thermo Fisher Scientific, Cat #13-6800; DARPP-32: 1:200, Cell Signaling, Cat #2306) for 1-1.5 h, followed by incubation with biotinylated secondary antibody (1: 200, Vector Laboratories, Cat #BA-2000; Cat #BA-1000) for 30 min, and then incubation with streptavidin conjugated Alexa 594/488 dye (1:1000, Thermo Fisher Scientific, Cat #S32356; Cat #S32354) or another 30 min. In double staining experiment, Alexa 488 conjugated secondary antibody (Thermo Fisher Scientific, Cat #A11008) was also used for detecting DARPP-32.
Both female and male mice were included for behavioral tests. There was no significant effect on gender for the behavioral tests. Motor activity was measured at 13 weeks of age in an open field. Mice were allowed to habituate to the experimental environment for at least one hour before assessment. Mice were placed in the center for 10 minutes, during which the distance travelled was recorded for the last 5 minutes by a digital camera coupled to the Smart Junior system. The distance travelled within each group was averaged separately. As for the clasping test, each mouse was suspended upside down by its tail for 15 seconds. They were administered three trials a week at 9, 11 and 13 weeks of age. The weekly percentage of limb clasping within each group was averaged separately. The weight of each mouse was recorded weekly.
Statistical analysis was carried out using GraphPad Prism 9 software. Results are presents as the mean±s.d. or ±s.e.m. Statistical significance of the mice Kaplan-Meier survival curves was determined by log-rank Mantel-Cox test.
This application claims the benefit of and priority to U.S. Application No. 63/489,732, filed Mar. 10, 2023, entitled “ALOX5-MEDIATED FERROPTOSIS ACTS AS A DISTINCT CELL DEATH PATHWAY UPON OXIDATIVE STRESS IN HUNTINGTON'S DISEASE” the contents of which is hereby incorporated by reference in its entirety.
This invention was made with government support under CA253059 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63489732 | Mar 2023 | US |
