Patent Application
20250034534
Hypophosphatasia (HPP) is an inborn error-of-metabolism caused by loss-of-function mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP is expressed in bones, teeth, liver, and kidney; its deficiency leads to mineralization defects caused by the accumulation of extracellular inorganic pyrophosphate (PPi), one of the major substrates of TNAP and a potent inhibitor of hydroxy apatite crystal formation and propagation. Murine studies have demonstrated that mineralizing skeletal and dental cells, including osteoblasts, chondrocytes, ameloblasts, odontoblasts, and cementoblasts, express TNAP.
In certain aspects, disclosed herein is a method of treating a subject with a soft bone disease comprising: administering a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue non-specific promotor to the subject in an intramuscular injection to a muscle, wherein administering the viral vector treats the soft bone disease. In some embodiments, the tissue non-specific promotor comprises a CAG promotor. In some embodiments, the viral vector comprises an adeno-associated vector. In some embodiments, the viral vector comprises an adeno-associated virus type 8 (AAV8) vector. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue non-specific alkaline phosphatase (TNAP). In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue non-specific alkaline phosphatase (TNAP) and a bone targeting sequence linked to the C-terminus of TNAP. In some embodiments, the bone targeting sequence is a deca-aspartate (D10) sequence. In some embodiments, the soft bone disease is hypophosphatasia (HPP). In some embodiments, the soft bone disease is hypophosphatasia (HPP), and wherein the hypophosphatasia is pediatric hypophosphatasia or infantile hypophosphatasia. In some embodiments, the soft bone disease is hypophosphatasia (HPP), and wherein the hypophosphatasia is late-onset hypophosphatasia. In some embodiments, the soft bone disease is caused by PHOSPHO1 deficiency. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse. In some embodiments, the mouse comprises a TNAP knockout mouse. In some embodiments, administering the viral vector results in increased plasma alkaline phosphatase (ALP) activity for at least two months. In some embodiments, following administering the viral vector, the viral vector is not detected in the brain. In some embodiments, following administering the viral vector, the viral vector is not detected in the gonads. In some embodiments, following administering the viral vector, the viral vector does not result in oncogenic effect in the subject. In some embodiments, the viral vector does not diffuse from the muscle.
In certain aspects, disclosed herein is a composition comprising a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue non-specific promotor. In certain aspects, disclosed herein is a composition for treating a soft bone disease, comprising a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue non-specific promotor, wherein the composition treats the soft bone disease and wherein the composition results in plasma ALP activity higher than in a subject not receiving the composition for at least eighteen months. In certain aspects, disclosed herein is a composition for treating a soft bone disease, comprising a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue non-specific promotor, wherein the composition treats the soft bone disease and wherein the viral vector does not diffuse from a target location. In some embodiments, the tissue non-specific promotor comprises a CAG promotor. In some embodiments, the viral vector comprises an adenoviral-associated virus. In some embodiments, the adenoviral-associated virus comprises an adeno-associated virus type 8 (AAV8) vector. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue non-specific alkaline phosphatase (TNAP). In some embodiments, the mineral-targeted alkaline phosphatase comprises a sequence for bone targeting linked to the C-terminus of TNAP. In some embodiments, the bone targeting sequence is a deca-aspartate (D10) sequence. In some embodiments, the soft bone disease is hypophosphatasia (HPP). In some embodiments, the hypophosphatasia is pediatric hypophosphatasia or infantile hypophosphatasia. In some embodiments, the hypophosphatasia is late-onset hypophosphatasia. In some embodiments, the soft bone disease is caused by PHOSPHO1 deficiency. In some embodiments, disclosed herein is a method of treating a soft bone disease, the method comprising administering the composition described herein to the subject in an intramuscular injection to a muscle.
In certain aspects, disclosed herein is a method of treating a subject with a dental disorder comprising: administering a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue non-specific promotor to the subject in an intramuscular injection, wherein administering the viral vector treats the dental disorder. In some embodiments, the tissue non-specific promotor comprises a CAG promotor. In some embodiments, the viral vector comprises an adenoviral-associated virus. In some embodiments, the viral vector comprises an adeno-associated virus type 8 (AAV8) vector. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue non-specific alkaline phosphatase (TNAP). In some embodiments, the mineral-targeted alkaline phosphatase further comprises a sequence for bone targeting linked to the C-terminus of TNAP. In some embodiments, the bone targeting sequence is a deca-aspartate (D10) sequence. In some embodiments, the dental disorder comprises at least one of a dentoalveolar disorder, teeth hypomineralization, and a periodontal disorder
All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
The patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition.
Asfotase alfa is a recombinant fusion protein comprising the TNAP ectodomain, a human IgG1 Fc domain for one-step purification, and a terminal deca-aspartate (D10) motif for mineral-targeting. In a murine model for infantile HPP, TNAP knockout (Akp2−/− or Alpl−/−) mice, treatment with daily subcutaneous injections of asfotase alfa preserved life span, improved skeletal phenotypes, and prevented epileptic seizures and dental defects. In humans, subcutaneous injections of asfotase alfa, three to seven times a week, in children or adults with HPP has demonstrated substantial and sustained efficacy with a good safety profile. Asfotase alfa saved lives of subjects with severe neonatal and infantile HPP and improved bone mineralization, motor function, and quality of life in subjects with adult HPP. At the same time, the patient burden of multiple injections per week to maintain the efficacy of asfotase alfa and the associated medical cost have prompted preclinical studies of alternative strategies for treating HPP.
A human chimeric recombinant alkaline phosphatase, ChimAP, and several forms of virus vectors expressing TNAP-D10 prolongs life, prevent seizures, and improve the skeletal phenotype of Alpl−/− mice. A single intravenous injection of a lentiviral or adeno-associated virus type 8 (AAV8) vector encoding TNAP-D10 leads to sustained correction of the skeletal phenotype of Alpl−/− mice, but the consequential wide distribution of vector genome to the whole body raised concern about possible transduction into germ cells.
Previous approaches to correct HPP-associated mineralization defects have met with mixed success or have had inherent limitations or drawbacks. ERT using asfotase alfa was shown to be very effective at correcting skeletal and dentoalveolar defects in Alpl−/− mice, with these translational studies leading to its approval in 2015 for treatment of patients with perinatal/infantile- and juvenile-onset HPP, except in Japan where asfotase alfa is approved for all ages. However, asfotase alfa ERT has a half-life of 2.28 days, requires multiple injections per week, is associated with injection site reactions, and is expensive, prompting additional preclinical studies of alternative strategies for HPP treatment. Daily subcutaneous injection of a soluble, non-mineral-targeting, recombinant chimeric alkaline phosphatase (ChimAP) prevented seizures, increased survival, but only partially improved the skeletal and dentoalveolar phenotypes in Alpl−/− mice. Studies using several different types of viral vectors with different modes of administration have been reported. Single intravenous injection of either lentiviral vector containing TNAP-D10 (HIV-TNAP-D10) or AAV8-TNAP-D10 resulted in sustained elevation of circulatory TNAP and phenotypic correction in Alpl−/− mice, but the viral sequence was detected in soft tissues, including the liver, lung, and heart, raising a safety concern for oncogenicity of the integrated lentiviral vector in these tissues. At the same time, intramuscular injection of scAAV8-MCK-TNAP-D10, a muscle-directed gene therapy consisted of a self-complementary type 8 AAV (scAAV8) vector and the muscle creatine kinase (MCK) promoter to limit the virus distribution, improved the skeletal phonotype in the Alpl−/− mice, but with limited elongation of the long bone and remaining hypomineralization. When compared to these previous methods, the approach described herein using a single intramuscular injection of AAV8-TNAP-D10, combines a more practical gene therapy, potentially improved safety profile due to limited tissue distribution, and yet remains highly effective at correcting mineralization defects.
Therapies for HPP other than asfotase alfa have been tested in a clinical setting without rigorous preclinical and translational studies. In a cross-sectional study that enrolled 51 patients with childhood and adult onset HPP, two out of four patients who were treated with teriparatide (parathyroid hormone 1-34) showed clinical and radiological improvement. In a phase IIA open-label study targeting eight adult patients with HPP, monoclonal antisclerostin antibody (BPS804) treatment resulted in increases in bone formation markers and bone mineral density. However, these anabolic agents have not been approved for HPP and an effective medical therapy for adult-onset HPP patients is needed. At the same time, a novel therapy with fewer injections may further benefit patients with childhood-onset HPP as well.
Concerns for AAV8-TNAP-D10 administration in human patients include continuously elevated circulatory ALP activity and its potential effects on PPi metabolism and development or aggravation of ectopic calcification. As described herein, a single intramuscular injection of AAV8-TNAP-D10 resulted in extremely high circulatory ALP activity, more than 500 times higher than that of WT mice. Extracellular PPi is known as a central regulator of biomineralization and is critical for controlling inappropriate soft tissue calcification in the body. Hydrolysis of PPi by TNAP-D10 may result in over-suppression of extracellular PPi in soft tissues. However, AAV8-TNAP-D10-treated Alpl−/− mice, with suppressed plasma PPi concentrations, did not develop any soft tissue calcifications during the observational window of 70 dpn. This contrasts with previous results from genetically modified mouse models with targeted overexpression of TNAP in the vasculature. TNAP overexpression in the smooth muscle cells using Tagln-Cre resulted in massive arterial calcifications in the ascending and descending aorta, carotid, and subclavian arteries. TNAP overexpression in endothelial cells using Tie2-Cre resulted in the partial calcification in the arteries of the heart, kidney, mesentery, pancreas, and spleen. These mouse models showed 20-30 times higher circulatory ALP activity while plasma PPi concentrations were similar to those of WT. From these results, one can assume that neither circulatory ALP activities nor circulatory PPi concentrations correlate with local PPi concentrations, which cannot be measured in vivo but presumably are low in target tissues with high TNAP expression. Additionally, the elevated urine PPi concentrations in TNAP-D10-treated Alpl−/− mice indicate that urine PPi metabolism is mostly dependent on TNAP expressed in the luminal surface of kidney proximal tubules, and independent of serum ALP activity and circulatory PPi concentrations. The qPCR analysis of the kidneys of treated mice indicated that genes for primary regulators of systemic PPi metabolism, including Ank, Enpp1, and Abcc6, were not affected by treatment.
Described herein is the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D10 for increasing lifespan and improving the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl−/−) mice, a murine model for severe infantile HPP. Alpl−/− mice received 3×1011 vector genomes/body of AAV8-TNAP-D10 within 5 days postnatal (dpn). AAV8-TNAP-D10 elevated serum ALP activity and suppressed plasma PPi. Treatment extended lifespan of Alpl−/− mice and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl−/− mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl−/− mice was similar to wild-type (WT), with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation, but reduced bone volume fraction in treated Alpl−/− mice versus controls. Treated Alpl−/− mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a potential alternative to ERT for the treatment of HPP.
Disclosed herein, in some embodiments, are methods useful for treating a subject in need thereof comprising administering a composition described herein. In some embodiments, the composition described herein comprises a viral vector described herein. In some embodiments, the disease is a soft bone disease, a dental disorder, or a combination thereof. In some embodiments, the method comprises administering a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue non-specific promotor to the subject in an intramuscular injection to a muscle.
In certain aspects, described herein is a method of administering a composition described herein to a subject in need thereof. In some embodiments, the methods improve at least one of the symptoms selected from the list consisting of life span, skeletal abnormalities, seizures, dental defects, bone mineralization, motor function, and quality of life. In some embodiments, the symptom is improved compared to a subject who has not received the treatment. In some embodiments, the symptom is improved compared to the subject prior to receiving the treatment.
In some embodiments, described herein is a method of increasing life span in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, described herein is a method of reducing skeletal abnormalities in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, described herein is a method of reducing seizures in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, described herein is a method of reducing dental defects in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, described herein is a method of increasing bone mineralization in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, described herein is a method of increasing motor function in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, described herein is a method of increasing quality of life in a subject in need thereof, the method comprising administering the compositions described herein to the subject. In some embodiments, the compositions comprise a viral vector for the delivery of an alkaline phosphatase.
HPP patients suffer from distinctive rickets and/or osteomalacia with a broad range of severity, as well as dental defects. There are seven major forms of HPP: life-threatening perinatal and infantile (OMIM #241500), benign perinatal, mild and severe childhood (OMIM #241510), adult (OMIM #136300), and odonto-HPP (OMIM #146300). Patients with perinatal HPP, the gravest form of HPP, often die in utero or soon after birth because of severe skeletal hypomineralization, respiratory failure due to thoracic cage dysplasia and hypoplastic lungs, and elevated intracranial pressure due to craniosynostosis. Dentoalveolar phenotypes, including premature exfoliation of primary teeth, periodontal disease, and enamel alternations, are commonly observed in patients with all forms of HPP.
In some embodiments, the subject has a soft bone disease. In some embodiments, the soft bone disease is hypophosphatasia (HPP). In some embodiments, the hypophosphatasia is pediatric hypophosphatasia or infantile hypophosphatasia. In some embodiments, the hypophosphatasia is late-onset hypophosphatasia.
In some embodiments, the disease comprises a dental disorder. All forms of HPP include dental involvement, with premature tooth loss being one of the most common manifestations. Characteristic dentoalveolar abnormalities observed in untreated Alpl−/− mice include inhibition of tooth root acellular cementum formation, PDL detachment, and enamel and dentin mineralization defects. As described herein, qualitative and quantitative analyses suggest that teeth and associated periodontal tissues significantly improved from AAV8-TNAP-D10-mediated gene therapy. AAV8-TNAP-D10-treated Alpl−/− mice showed normal formation and mineralization of molar and incisor enamel and dentin, and the restoration of acellular cementum, PDL cellularity, and PDL-cementum attachment. Treated Alpl−/− mice also showed well-developed alveolar bone with mildly reduced mineral density. In some embodiments, the dental disorder comprises at least one of a dentoalveolar disorder, teeth hypomineralization, and a periodontal disorder.
There are more than 400 mutant alleles identified for the ALPL. The inheritance pattern of perinatal and infantile HPP is often autosomal recessive, with most patients being compound heterozygotes for pathogenic ALPL mutations that result in almost null ALP activity, but some are homozygous for recessive alleles and most adult and odonto-HPP patients harbor a single dominant-negative ALPL allele. In some embodiments, the subject has a mutation in the ALPL gene. In some embodiments, the subject has a PHOSPHO1 deficiency.
In some embodiments, the viral vector is administered at least once. In some embodiments, administering the viral vector results in increased plasma alkaline phosphatases (ALP) activity. In some embodiments, ALP activity is increased for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments, ALP activity is increased for at least 1 week. In some embodiments, ALP activity is increased for at least 2 weeks. In some embodiments, ALP activity is increased for at least 3 weeks. In some embodiments, ALP activity is increased for at least 3 weeks. In some embodiments, ALP activity is increased for at least 4 weeks. In some embodiments, ALP activity is increased for at least 2 months. In some embodiments, ALP activity is increased for at least 3 months. In some embodiments, ALP activity is increased for at least 4 months. In some embodiments, ALP activity is increased for at least 5 months. In some embodiments, ALP activity is increased for at least 6 months.
In some embodiments, the viral vector is administered intramuscularly. In some embodiments, the viral vector does not diffuse away from the site of injection. In some embodiments, the viral vector does not diffuse away from the muscle. In some embodiments, after administration of the viral vector, the viral vector is not detected in the brain, gonads, or a combination thereof. In some embodiments, after administration of the viral vector, the viral vector is not detected in the brain. In some embodiments, after administration of the viral vector, the viral vector is not detected in the gonads.
In patients with HPP, with or without treatment, several types of ectopic calcifications are commonly observed: ocular calcification, nephrocalcinosis, and painful periarthritis. In some embodiments, following administering the viral vector, the subject does not develop an ectopic calcification. In some embodiments, the ectopic calcification is selected from the list of ocular calcification, nephrocalcinosis, painful periarthritis, or kidney stones. In some embodiments, the subject does not develop hypercalcemia. In some embodiments, the subject does not develop hypercalciuria. In some embodiments, the subject does not develop ectopic calcifications in a tissue of the subject. In some embodiments, the tissue is selected from the list consisting of kidneys, aorta, coronary arteries, and brain. In some embodiments, following administering the viral vector, the viral vector does not result in oncogenic effect in the subject.
In certain aspects, described herein are compositions for delivery of a therapeutic peptide to a subject in need thereof. In some embodiments, described herein is a viral vector for the delivery of an alkaline phosphatase to a subject in need thereof. In some embodiments, the viral vector comprises the sequence of a therapeutic peptide. In some embodiments, the therapeutic peptide comprises an alkaline phosphatase. In some embodiments, the therapeutic peptide comprises a mineral-targeted alkaline phosphatase. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue non-specific alkaline phosphatase (TNAP).
In some embodiments, the alkaline phosphatase comprises an alkaline phosphatase comprising the sequence of SEQ ID NO: 15. In some embodiments, the alkaline phosphatase comprises an alkaline phosphatase with at least 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or 100% sequence identity with SEQ ID NO: 15.
In some embodiments, the mineral-targeted alkaline phosphatase comprises a sequence for bone targeting. The sequence for bone targeting may be linked to the C-terminus of the mineral-targeted alkaline phosphatase. The sequence for bone targeting may be linked to the N-terminus of the mineral-targeted alkaline phosphatase. The bone-targeting sequence may comprise at least one aspartate. The bone-targeting sequence may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more aspartates. In some embodiments, the bone-targeting sequence comprises a deca-aspartate (D10) sequence. In some embodiments, the bone targeting sequence comprises the sequence DDDDDDDDDD (SEQ ID NO: 17). In some embodiments, the bone-targeting peptide comprises a repeat sequence of aspartate-serine-serine (DSS, SEQ ID NO: 18). In some embodiments, the bone-targeting sequence comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats of aspartate-serine-serine. In some embodiments, the bone-targeting sequence comprises the sequence KRRTPVRE (SEQ ID NO: 19). In some embodiments, the bone-targeting sequence comprises the sequence KNFQSRSH (SEQ ID NO: 20). In some embodiments, the bone-targeting sequence comprises the sequence KTYASMQW (SEQ ID NO: 21). In some embodiments, the bone-targeting sequence comprises at least one sequence selected from the list consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
The peptides described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors and the like. In the expression vectors regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes.
In some embodiments, the viral vector comprises a non-tissue specific promoter. In some embodiments, the tissue non-specific promotor comprises a CAG promotor. In some embodiments, the viral vector comprises a promoter with the sequence of SEQ ID NO 16. In some embodiments, the viral vector comprises a non-tissue specific promoter with at least with at least 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or 100% sequence identity with SEQ ID NO. 16.
The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed. In some embodiments, the vector comprises an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 vector. In some embodiments, the vector is an AAV8 vector.
Plasmid vectors can be linearized for integration into a chromosomal location. Vectors can comprise sequences that direct site-specific integration into a defined location or restricted set of sites in the genome (e.g., AttP-AttB recombination). Additionally, vectors can comprise sequences derived from transposable elements.
The compositions disclosed herein are formulated in any suitable manner for administration. Any suitable technique, carrier, and/or excipient is contemplated for use with the compositions disclosed herein. Non-limiting examples of cosmetic, dermatological, or pharmaceutically acceptable carriers and excipients suitable for formulation can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; Pharmaceutical Dosage Forms and Drug Delivery Systems, Eighth Ed. (Lippincott Williams & Wilkins 2004); and Muller, R. H., et al., Advanced Drug Delivery Reviews 59 (2007) 522-530, each of which is incorporated by reference in its entirety.
In some embodiments, the pharmaceutically acceptable carriers or excipients disclosed herein include, but are not limited to one or more: pH modifying agent (e.g., buffering agents), stabilizing agents, thickening agents, colorant agents, preservative agents, emulsifying agents, solubilizing agents, antioxidant agents, or any combination thereof. Other suitable compounds contemplated herein and within the knowledge of a practitioner skilled in the relevant art are found in the Handbook of Pharmaceutical Excipients, 4th Ed. (2003), the entire content of which is incorporated by reference herein.
In some embodiments, the compositions disclosed herein comprise one or more preservatives. The preservative, when utilized, is in an amount sufficient to extend the shelf-life or storage stability, or both, of the topical formulations disclosed herein. Exemplary preservatives include, but are not limited to: tetrasodium ethylene-diamine tetraacetic acid (EDTA), methyl, ethyl, butyl, and propyl parabens, benzophenone-4, methylchloroisothiazolinone, methylisothiazolinone, sodium benzoate, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenylethylalcohol, dehydroacetic acid, sorbic acid, benzalkonium chloride (BKC), benzethonium chloride, phenol, phenylmercuric nitrate, and thimerosal.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not intended to be limited solely to the recited items. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
The terms “effective amount” or “therapeutically effective amount,” as used herein, generally refer to a sufficient amount of an agent or a compound which will relieve, to some extent, or reduce the likelihood of the occurrence of one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The terms “effective amount” or “therapeutically effective amount” typically include, for example, a prophylactically effective amount. For example, a “prophylactically effective amount” is the amount of the composition described herein that is required to reduce the risk of absorption, transmission, or function of a pathogen in an individual or transmission of a pathogen to another individual.
The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject can be a mouse. The subject can be a TNAP knockout mouse. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
The terms “about” or “approximately,” as used herein, generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part upon how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 10% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold of a value.
The following examples are illustrative and non-limiting to the scope of the compositions, methods, and formulations described herein.
TNAP knockout (Alpl−/−) mice were created by inserting a Neo cassette into exon 6 of the mouse Alpl gene via homologous recombination. (Narisawa S, Frohlander N, Millan J L. Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn. March 1997; 208(3):432-46.) Alpl−/− mice phenocopy human infantile HPP, showing normal appearance and being indistinguishable from other siblings at birth. They displayed almost zero circulatory ALP activity, develop epileptic seizures, become cachectic, and die by 10 to 12 days postnatal (dpn) without additional supportive treatment. Alpl−/− mice were maintained in a 12.5% C57Bl/6 and 87.5% 129J background and genotyped by PCR using genomic DNA extracted from toe samples within five days after birth. All animals (breeders, nursing mothers, pups, and weanlings) in this study were given free access to regular diet (2018 Teklad global 18% protein extruded rodent diets or 2019 Teklad global 19% protein rodent diets, Envigo, Indianapolis, IN, USA) with a standard level of vitamin B6, increased level of which was reported to improve the lifespan of Alpl−/− mice. The institutional Animal Care and Use Committee (IACUC) approved all the animal studies.
Virus Vector Encoding the Human TNAP-D10 cDNA
TNAP-D10 contains recombinant human soluble TNAP (sALP), and a deca-aspartate (D10) sequence at the C terminus, which enables TNAP to target mineralized tissues, such as bone and teeth. The human IgG1 Fc domain present in asfotase alfa to enable a one-step purification is absent from the product of this vector as purification is not required for viral-vector mediated in vivo expression. Recombinant AAV type 8 vector encoding TNAP-D10 (AAV8-TNAP-D10) under control of the CAG promoter was generated using the HEK293 cell line by the triple transfection method, purified, and then titrated as previously reported. Recombinant AAV type 8 vector encoding GFP (AAV8-GFP) was used as a control. Mice received a single injection of AAV8-TNAP-D10 at a dose of 3×1011 vector/genomes (vg)/body into the quadriceps femoris within 5 dpn. Eleven Alpl−/− mice (male n=6, female n=5) and 14 wild type (WT) controls (male n=7, female n=7) were included in this study. After genotyping, all the Alpl−/− mice received a single injection of AAV8-TNAP-D10 at a dose of 3×1011 vector genomes (vg)/body into the quadriceps femoris within 5 dpn. Three control WT mice received the same dose of AAV8-TNAP-D10 to assess its effect on PPi metabolism and soft tissue calcification when endogenous TNAP activity is present. Seven WT mice received the same amount of control AAV8-GFP vector and four WT mice were untreated; their data were combined and analyzed together as WT, due to lack of substantial differences between them. Mice were euthanized at 70 dpn by exsanguination, after intraperitoneal administration of Avertin. In mice, 70 dpn is the end of puberty, equivalent to a human age of 20 years, and root formation and cellular cementum formation have been completed. For the analysis of untreated Alpl−/−, 17 WT, 7 heterozygote (Alpl+/−), and 10 Alpl−/− pups were collected at 10 dpn, euthanized by exsanguination after intraperitoneal administration of Avertin.
Body weight of the mice was measured at 35 and 70 dpn. Blood was collected from the orbital sinus of isoflurane-anesthetized mice using Pasteur pipets every 4 weeks after injection. Spot urine samples were collected simultaneously.
Blood was collected into two types of BD Microtainers coated with either clot activator or lithium heparin (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Blood for plasma collection and urine samples were placed on ice. Microtainers were then spun at 7,000 g for 10 minutes. Twenty μL of heparin-plasma was deproteinized using a Microcon-10 kDa Centrifugal Filter Unit with Ultracel-10 membrane (MilliporeSigma, Merck KGaA, Darmstadt, Germany), centrifuged at 14,000 g for 20 minutes. Urine was diluted 1:3 with 10 mM HEPES. Samples were stored at −80° C. for further analyses.
Plasma PPi concentration was measured according to the protocol described previously. Five μL of deproteinized plasma sample and the PPi standard ranging from 0.125 μM to 20 μM (Sodium pyrophosphate decahydrate, Sigma-Aldrich, St. Louis, MO, USA) was added to 45 μL of assay mixture containing 90 μM adenosine 5′ phosphosulfate sodium salt (APS) (Sigma-Aldrich), 22.5 μM MgCl2, 11.25 mM HEPES with 0.9 U/mL recombinant Yeast ATP-sulfurylase/MET3 (R&D Systems, Inc., Minneapolis, MN, USA). The mixture was incubated at 37° C. for 30 minutes and heat-inactivated at 90° C. for 10 minutes. Ten μL of each sample were then transferred into a 96-well white-bottom plate and mixed with 50 μl of BacTiter-Glo Microbial Cell Viability Assay (Promega Corporation, Madison, WI, USA). Luminescence was measured by FilterMax F5 Multimode Microplate Readers (Molecular Devices, LLC., San Jose, CA, USA).
Serum ALP activity was measured using an enzymatic assay. Five μL of serum and recombinant human ALP standards (0.099 to 216.0 ng/mL) were mixed with 95 μL of 10 mM pNPP in diethanolamine (DEA) buffer (pH 9.8) containing 1.0 mM MgCl2 and 20 μM ZnCl2. The increase in A405 nm was measured using OptiMax Microplate Absorbance Reader (Molecular Devices, LLC., San Jose, CA, USA) for 15 minutes. QuantiChrom Calcium Assay Kit, QuantiChrom Creatinine Assay Kit, and QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA, USA) were used to measure serum and urine calcium, urine creatinine, and serum urea concentrations, respectively. Stanbio Phosphorus Liqui-UV (EKF Diagnostics-Stanbio Laboratory, Boerne, TX, USA) was used to measure serum and urine phosphorus concentrations.
Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from the kidney using RNAeasy Plus Kit (Qiagen LLC, Germantown, MD, USA), and reverse transcription was carried out using PrimeScript RT Master Mix (Takara Bio USA, Inc., Mountain View, CA, USA). Real-time qPCR was performed in a 384-well plate in an Applied Biosystems 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using cDNA equivalent to 25 ng total RNA and DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was run for 40 cycles at an initial temperature of 95° C. for 7 min and then at 95° C. for 10 s followed by 60° C. for 15 s. Ct values were determined by the software and the amplification of the target gene was normalized to that of 18S ribosomal RNA (Rn18s). Sequences of the primer pairs used for PCR are as follows: Alpl (NM_007431.3) F-CTGCCACTGCCTACTTGTGT (SEQ ID NO: 1) and R-GATGGATGTGACCTCATTGC (SEQ ID NO: 2); Ank (NM_020332.4) F-CTGCTGCTACAGAGGCAGTG (SEQ ID NO: 3) and R-GACAAAACAGAGCGTCAGCGA (SEQ ID NO: 4); Enpp1 (NM_001308327.1) F-TCACGCCACCGAGACTAAATA (SEQ ID NO: 5) and R-TGCAGTAGGGTGTCATGAAGG (SEQ ID NO: 6), Abcc6 (NM_018795.2) F-CATCTTGCCAGGAATCAACACT (SEQ ID NO: 7) and R-ACCAGGGACAAGCACAGGTA (SEQ ID NO: 8); Il6 (Interleukin 6) (NM_031168.2) F-CAAAGCCAGAGTCCTTCAGAGAG (SEQ ID NO: 9) and R-TTAGCCACTCCTTCTGTGACTCC (SEQ ID NO 10); Tnf (TNF-alpha) (NM_013693.3) F-CAGCCTCTTCTCATTCCTGCT (SEQ ID NO: 11) and R-GCCATTTGGGAACTTCTCATC (SEQ ID NO: 12); Rn18s (NR_003278.3) F-TTGATTAAGTCCCTGCCCTTTGT (SEQ ID NO: 13) and R-CGATCCGAGGGCCTCACTA (SEQ ID NO: 14).
Radiographic images of entire skeletons and forelimbs, hindlimbs, and skulls were obtained with a Faxitron MX-20DC4 (Chicago, IL, USA), using energy of 20 kV. Lengths of the femur, tibia, humerus, and radius were measured using ImageJ (Rasband, W. S., ImageJ, National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2018). Head measurements were performed using the following landmarks: (Liu J, Nam H K, Wang E, Hatch N E. Further analysis of the Crouzon mouse: effects of the FGFR2(C342Y) mutation are cranial bone-dependent. Calcif Tissue Int. May 2013; 92(5):451-66) nose length, the length from the rostal point of intersection of nasal bones to the caudal point of intersection of nasal bones; cranial length, the length from the rostal point of intersection of nasal bones to the median (midline) point of the posterior margin of the foramen magnum; cranial width, the length from the right joining of squamosal body to zygomatic process of squamous portion of temporal bone to the left counterpart.
After fixation in 4% paraformaldehyde/PBS solution, hemi-mandibles and femurs were scanned in a μCT 50 scanner (ScancoMedical, Bassersdorf, Switzerland) at 70 kV, 76 μA, 0.5 Al filter, 900 ms integration time, and 6 or 10 μm voxel dimension for mandibles and femurs, respectively. Reconstructed images were calibrated to 5 known densities of hydroxyapatite and analyzed using AnalyzePro (version 1.0; AnalyzeDirect, Overland Park, KS). For femurs, trabecular and cortical bones were segmented at 350 and 650 mgHA/cm3, respectively. The trabecular bone was traced using 50 slices (total of 0.5 mm) proximal to the distal femur growth plate to quantify bone volume (BV), total volume (TV), bone volume fraction (Tb. BV/TV), trabecular number (Tb. N), thickness (Tb. Th), spacing (Tb. Sp), connective density (1/mm3) and mineral density (Tb. BMD). For the cortical bone, 50 slices of the mid-femur of each bone was used to quantify cortical bone volume fraction (Ct. BV/TV), cortical thickness (Ct. Th), porosity, and mineral density (Ct. BMD).
The first mandibular molar and associated alveolar bone was quantitively analyzed as previously described. The alveolar bone region of interest (ROI) included the area between 240 μm mesial to the most mesial point of the first molar mesial root and 240 μm distal to the most distal point of the distal root. Enamel was segmented above 1,600 mg HA/cm3, while dentin/cementum and alveolar bone were segmented at 550-1,600 mg HA/cm3.
Skeletal and soft tissues were fixed in 4% paraformaldehyde/PBS solution and processed for histological analyses. Undecalcified fixed bone samples were placed in 30% sucrose/PBS solution and then cryo-embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Torrance, CA, USA) in hexane dry ice bath and were sectioned by the Kawamoto method. (Kawamoto T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol. May 2003; 66(2):123-43) The tibial and femur bones were placed in 0.125M EDTA/10% formalin (pH 7.3) solution for 7 days for decalcification and were then paraffin embedded. Soft organs were either paraffin embedded or embedded in an OCT in an ethanol dry ice bath. Hematoxylin and eosin (H&E), von Kossa, von Kossa/van Gieson, and Safranin O staining were performed according to standard methods. Tissue ALP activity was assayed by incubating the OCT-embedded sections in freshly mixed substrate solution made of one volume of 0.2 mg of Naphthol AS-MX phosphate disodium salt per ml of water and one volume of 1.2 mg FAST Violet B salt per mL of 0.2 M Tris-HCl (pH 8.9) at room temperature for 60 minutes, and counterstaining in methyl green solution. Slides were observed under IX81 Olympus Microscope (Olympus Corporation, Center Valley, PA, USA) or scanned by Aperio AT2 system (Leica Biosystems of Leica Microsystems Inc., Buffalo Grove, IL, USA). Bone volume fraction (BV/TV) was measured using ImageJ.
Left hemi-mandibles were fixed in Bouin's solution overnight, decalcified in an acetic acid/formalin/sodium chloride solution, processed for paraffin embedding and sectioned at 5 μm thickness in the coronal plane. Paraffin sections were stained with H&E to assess tooth and associated periodontium. Immunohistochemistry (IHC) procedures were performed as described previously. (Foster B L, Ao M, Salmon C R, Chavez M B, Kolli T N, Tran A B, et al. Osteopontin regulates dentin and alveolar bone development and mineralization. Bone. February 2018; 107:196-207.) Primary antibodies included: monoclonal rat anti-human alkaline phosphatase IgG (TNAP) (R&D systems, Minneapolis, MN, USA); polyclonal rabbit anti-mouse bone sialoprotein (BSP) IgG; and polyclonal LF-175 rabbit anti-mouse osteopontin (OPN) IgG. In situ hybridization was performed for mouse Alpl with (RNAscope 2.5 HD Detection reagent kit-RED assay, Advanced Cell Diagnostics) following the manufacturer's instructions as previously described. (Zhang H, Chavez M B, Kolli T N, Tan M H, Fong H, Chu E Y, et al. Dentoalveolar Defects in the Hyp Mouse Model of X-linked Hypophosphatemia. J Dent Res. April 2020; 99(4):419-28) For acellular cementum/PDL analysis, H&E-stained images captured with the same acquisition parameters were segmented using the color map function (5 Ramps) in ImageJ. This method is to pseudo-color the images to make differences between pixel values more apparent for improved tissue visualization. The values for acellular cementum and mantle dentin thickness represent the average of three linear measurements were taken at 90 μm, 100 μm, and 110 μm from the cemento-enamel junction (CEJ) using the ImageJ straight-line function. Mantle dentin is the outer layer, less mineralized dentin adjacent to acellular cementum. For assessing cellularity, a region of 5.5 mm2 area was defined 100 μm apical to the CEJ for counting cells in the PDL space.
All the statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, California, USA). Data are expressed as mean±standard deviation (SD) in charts. One-way analysis of variance (ANOVA) followed by Turkey's multiple comparisons test was performed to compare the differences among control WT, treated Alpl−/−, and treated WT mice. Unpaired t-test with Welch's correction was performed to compare each gene expression in the kidneys of WT and treated Alpl−/− mice. Significance was determined by P<0.05 and shown in the charts as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
Improved Survival and Correction of Plasma PPi in AAV8-TNAP-D10-Treated Alpl−/− Mice
While untreated Alpl−/− pups die within 10 to 12 days after birth, all Alpl−/− mice treated with AAV8-TNAP-D10 in this study did not develop epileptic seizures and were viable until the endpoint of the study at 70 dpn. The untreated Alpl−/− pups did not feed well and were significantly smaller than WT littermates at 10 dpn (
Serum ALP activity was significantly higher in Alpl−/− and WT mice injected with AAV8-TNAP-D10, approximately 500-800 times higher than that of control WT mice (
0.081 ± 0.080b
aP < 0.0001 compared to WT,
bP < 0.05 compared to WT,
cP < 0.0001 compared to Alpl−/− + TNAP
Improved Bone Microstructure in AAV8-TNAP-D10-Treated Alpl−/− Mice
In previous studies, untreated 20-23 dpn Alpl−/− mice demonstrated profound skeletal abnormalities including reduced tissue mineral density and bone fractures. Radiography of 70 dpn Alpl−/− mice treated with AAV8-TNAP-D10 revealed grossly normal skeletal development comparable to WT littermates (
Three-dimensional μCT renderings of AAV8-TNAP-D10-treated Alpl−/− femurs showed bone morphology similar to WT femurs; however, the bone was ˜7-9% shorter in treated Alpl−/− females compared with WT controls (
Von Kossa/van Gieson staining of femurs and lumbar spines showed no measurable osteoid surface (
AAV8-TNAP-D10 Prevents HPP-Associated Dentoalveolar Defects in Alpl−/− Mice
Data from male and female dentoalveolar tissues were combined together due to lack of substantial sex-related differences in previous reports of HPP mouse models, and because sex-related trends in these data were not found. Compared to untreated and treated WT controls, first mandibular molars and surrounding alveolar bone of AAV8-TNAP-D10-treated Alpl−/− mice appeared grossly normal (
Histology of AAV8-TNAP-D10-treated Alpl−/− mouse mandibles revealed normal tooth structures largely indiscernible from those of WT controls, with similar morphology, tissue organization, presence of acellular cementum on root surfaces, and periodontal attachment (
Virus Vector Encoding the Human TNAP-D10 cDNA
Recombinant adeno-associated virus vector serotype 8 (AAV8) encoding mineral-targeted TNAP (AAV8-TNAP-D10) was generated using the HEK293 cell line. Recombinant AAV8 vector encoding GFP (AAV8-GFP) was used as a control. Eight weeks old AlplPrx1/Prx1 and WT siblings mice, as well as Phospho1−/− and WT littermates, received a single intramuscular injection of AAV8-TNAP-D10 or AAV8-GFP vector as control at a dose of 3×1011 vector genomes (vg)/body into the quadriceps femoris.
Eight weeks old AlplPrx1/Prx1 and WT siblings mice were fed with a diet containing adenine (0.2%) for four weeks, and adenine+high phosphorus diet (0.2% adenine and 1.8% phosphorus) for another 4 weeks, protocol adapted from Tani et al., Inhibition of tissue-nonspecific alkaline phosphatase protects against medial arterial calcification and improves survival probability in the CKD-MBD mouse model. J Pathol. January 2020; 250(1):30-41, to induce a state of uremia that recapitulates CKD, which leads to vascular calcification. The animals were monitored weekly by measuring body weight and observing clinical signs such as posture and gait, difficulty of eating, etc. Animals received a single dose of AAV8-TNAP-D10 or AAV8-GFP treatment on the same day as the induction of uremia. After 8 weeks, mice were sacrificed, and soft organs such as kidneys, aorta, and heart, were harvested from each animal to assess the status of calcification and whether the treatment can worsen the ectopic calcification.
Body weight of the mice was first measured, and then they were anesthetized with isoflurane for blood (plasma/serum) collection from the orbital sinus of mice using Pasteur pipets before the treatment and 60 days after injection for late-onset HPP mice, and after 45 and 90 days of treatment for the pseudo-HPP mice. Blood samples were centrifuged at 7,500 rpm for 10 minutes. For PPi analysis, twenty μL of heparin-plasma was deproteinized using a Microcon-10 kDa Centrifugal Filter Unit with Ultracel-10 membrane (MilliporeSigma, Merck KGaA, Darmstadt, Germany), centrifuged at 14,000 g for 20 minutes. Samples were stored at −80° C. for further analyses.
Plasma PPi concentrations were measured. Five μL of deproteinized plasma sample and the PPi standard ranging from 0.125 μM to 20 μM (Sodium pyrophosphate decahydrate, Sigma-Aldrich, St. Louis, MO, USA) was added to 45 μL of assay mixture containing 90 μM adenosine 5′ phosphosulfate sodium salt (APS) (Sigma-Aldrich), 22.5 μM MgCl2, 11.25 mM HEPES with 0.9 U/mL recombinant Yeast ATP-sulfurylase/MET3 (R&D Systems, Inc., Minneapolis, MN, USA). The mixture was incubated at 37° C. for 30 minutes and heat-inactivated at 90° C. for 10 minutes. Ten μL of each sample were then transferred into a 96-well white-bottom plate and mixed with 50 μL of BacTiter-Glo Microbial Cell Viability Assay (Promega Corporation, Madison, WI, USA). Luminescence was measured by FilterMax F5 Multimode Microplate Readers (VWR International, LLC., Radnor, PA, USA). Serum ALP activity was measured using an enzymatic assay. Five μL of serum and recombinant human ALP standards (0.099 to 216.0 ng/mL) were mixed with 95 μL of 10 mM pNPP in diethanolamine (DEA) buffer (pH 9.8) containing 1.0 mM MgCl2 and 20 μM ZnCl2. The absorbance of the kinetics assay was measured at 405 nm using OptiMax Microplate Absorbance Reader (Molecular Devices, LLC., San Jose, CA, USA) for 15 minutes. QuantiChrom Calcium Assay Kit and QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA, USA) were used to measure serum calcium, and serum urea concentrations, respectively. Stanbio Phosphorus Liqui-UV (EKF Diagnostics-Stanbio Laboratory, Boerne, TX, USA) was used to measure serum phosphorus concentrations.
Radiographic images of the whole skeleton as well as the isolated bones such as the skull, vertebra, long bones, and hemi-mandibles, were obtained with the Faxitron MX-20DC4 radiograph system (Chicago, IL, USA), using the energy of 20 kV.
Before micro-CT analysis, long bones and right hemimandibles were fixed in 4% paraformaldehyde/PBS solution. Femurs were scanned using a Sky Scan 1172 scanner (Bruker Micro-CT, Kontich, Belgium) at 55 kV, 181 μA, 0.5 mm Al filter, 280 ms integration time, and m voxel dimension. Micro-CT images were reconstructed in NRecon software, and reconstructed images were calibrated to three known densities of hydroxyapatite and analyzed using AnalyzePro (version 1.0; AnalyzeDirect, Overland Park, KS). Trabecular and cortical bones of femurs were segmented at 400 and 550 mgHA/cm3, respectively. Micro-CT analysis of femurs was performed. Briefly, the trabecular bone was traced using 50 slices (total of 0.5 mm) proximal to the distal femur growth plate to quantify bone volume (BV), total volume (TV), bone volume fraction (Tb. BV/TV), trabecular number (Tb. N), thickness (Tb. Th), spacing (Tb. Sp), connective density (1/mm3), and mineral density (Tb. BMD). For the cortical bone, 50 slices of the mid-femur of each bone were used to quantify cortical bone volume fraction (Ct. BV/TV), cortical thickness (Ct. Th), marrow area, and mineral density (Ct. BMD).
Hemimandibles were scanned using a micro-CT 50 scanner (Scanco Medical, Bassersdorf, Switzerland) at 70 kV, 76 μA, 0.5 Al filter, 900 ms integration time, and 6 μm voxel dimension. Reconstructed images were calibrated to five known densities of hydroxyapatite and analyzed using AnalyzePro (version 1.0; AnalyzeDirect, Overland Park, KS). Micro-CT analysis of teeth was performed using the first mandibular molar and associated alveolar bone. The alveolar bone region of interest was defined to include 240 μm mesial to the most mesial point of the first molar mesial root and 240 μm distal to the most distal point of the distal root. Alveolar bone and dentin/cementum were segmented at 650-1,600 mg/cm3 HA, while enamel was segmented above 1,600 mg/cm3 HA.
Treated AlplPrx1/Prx1 and Phospho1−/− mice were euthanized 60 days and 90 days, respectively, by exsanguination after an intraperitoneal administration of Avertin, and skeletal/dental tissues were collected, fixed in 4% paraformaldehyde/PBS or Boiun's solution, and processed for histological analysis. The long bones and vertebrae were placed in 0.125M EDTA/10% formalin (pH 7.3) solution for 7 days for decalcification and then were paraffin-embedded and sectioned at 5 μm thickness. Hemi-mandibles were fixed in Bouin's solution, decalcified in an acetic acid/formalin/sodium chloride solution, processed for paraffin embedding, and sectioned at 5 μm thickness. Hematoxylin and eosin (H&E) and alizarin red staining were performed according to standard methods. Slides were scanned by the Aperio AT2 system (Leica Biosystems of Leica Microsystems Inc., Buffalo Grove, IL, USA).
All statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, California, USA) using one-way ANOVA followed by Tukey's multiple comparisons test for comparisons with more than two groups or Unpaired t-test for comparisons between two groups. Values were expressed as the mean±SD. Differences were statistically significant at *p<0.05.
Body weight and biochemical markers in serum and/or plasma were monitored from adult HPP and WT mice prior to treatment and 60 days after the AAV8-TNAP-D10 injection. Body weights of female adult HPP mice were slightly lower than WT littermates prior to the injection. After 60 days of treatment, there was an increase in body weight while in males no differences were observed. Male adult HPP mice showed body weight increase after AAV8-TNAP-D10 treatment when compared to HPP AAV8-GFP injected mice (
nsP > 0.05—Not significant.
After 60 days of administering AAV8-TNAP-D10, serum ALP activity was significantly higher in female and male adult HPP mice and in WT mice compared to the vehicle (AAV8-GFP) group (
nsP > 0.05 - Not significant.
Biochemical markers were assessed in Phospho1−/− mice 45 and 90 days after AAV8-TNAP-D10 treatment. Serum ALP activity was significantly higher and plasma PPi levels were lower in both female and male AAV8-TNAP-D10 treated mice compared to AAV8-GFP treated mice (
Improvements in the Skeleton with AAV8-TNAP-D10 Treatment without Evidence of Ectopic Calcification in Soft Organs
Radiographic images of female and male adult HPP mice revealed normal skeletal development when compared to WT littermates (
Micro-CT analysis of bone parameters showed that untreated HPP mice had a statistically significant reduction in cortical bone volume fraction (Ct. BV/TV) compared with untreated and AAV8-TNAP-D10 treated WT littermates. The reduced cortical BV/TV in untreated HPP mice was associated with a significantly reduced cortical thickness (Ct. Th) and increased marrow area (Ma. Ar) vs. untreated and treated WT littermates. However, the cortical bone mineral density (Ct. BMD) of untreated HPP mice was not statistically significant different from treated and untreated WT littermates. Gene therapy using the AAV8-TNAP-D10 vector significantly improved cortical bone in HPP mice, including bone fraction (Ct. BV/TV), thickness, and marrow area. Notably, no statistically significant difference was observed between treated and untreated WT mice, suggesting no adverse effects of AAV8-TNAP-D10 vector-mediated gene therapy over 60 days (
Radiographic images of female and male Phospho1−/− mice showed apparent normal skeletal development, except for a spinal deformity, i.e., the abnormal moderate and severe degrees of scoliosis in vertebrae of untreated Phospho1−/− mice. (
In addition, the treatment with the AAV8-TNAP-D10 vector did not promote ectopic calcification in the kidney, heart, or aorta of female and male adult HPP mouse model (
Micro-CT analysis of the first mandibular molar and associated alveolar bone showed that adult HPP mice had no evident defects in enamel, dentin/cementum, bone volumes, and mineral densities, compared with untreated WT or AAV8-TNAP-D10 treated WT and HPP mice. However, there was a statistically significant difference in the enamel volume between AAV8-TNAP-D10 treated WT and untreated HPP. Similarly, micro-CT analysis of continuously erupting incisors showed no significant difference in enamel and dentin volumes and densities. Together, these results suggest that Alpl ablation in Prx1-expressing dental mesenchymal cells may be dispensable for dentoalveolar formation and mineralization. Additionally, AAV8-TNAP-D10-mediated gene therapy for 60 days had no adverse effects as no difference was observed between treated and untreated WT teeth. Histological analysis of untreated HPP teeth revealed no morphological differences compared with untreated WT or both AAV8-treated-HPP and WT littermates. No obvious difference in tissue organization, periodontal attachments, or acellular cementum between treated and untreated HPP (
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Patent Application No. 63/193,366, filed May 26, 2021, which application is entirely incorporated herein by reference.
This invention was made with government support under Grant No. DE012889 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/030928 | 5/25/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63193366 | May 2021 | US |
