US20170189488A1 - Codon-optimized recombinant plasmid, method of stimulating peripheral nerve regeneration, and method of treating nerve damage in humans - Google Patents

Codon-optimized recombinant plasmid, method of stimulating peripheral nerve regeneration, and method of treating nerve damage in humans Download PDF

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US20170189488A1
US20170189488A1 US15/460,668 US201715460668A US2017189488A1 US 20170189488 A1 US20170189488 A1 US 20170189488A1 US 201715460668 A US201715460668 A US 201715460668A US 2017189488 A1 US2017189488 A1 US 2017189488A1
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vector
vegf
fgf2
nervous system
growth factor
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Artur Aleksandrovich Isaev
Albert Anatolyevich Rizvanov
Ruslan Faridovich Masgutov
Aleksei Andreevich Bogov
IInur lldusovich Salafutdinov
Roman Vadimovich Deev
llya Yadigerovich Bozo
Igor Leonidovich Plaksa
Andrei Alekseevich Bogov
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NextGen Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • C12N15/09—Recombinant DNA-technology
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    • C12N2810/00—Vectors comprising a targeting moiety

Definitions

  • the present invention is in the field of medicine and more specifically in the fields of neurosurgery, traumatology and maxillofacial surgery as applied to treatment of peripheral nerve injuries. These injuries are effectively treated with engineered recombinant nucleic acids.
  • engineered recombinant nucleic acid is a plasmid that encodes and expresses vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF2) when contacted with or transformed into a tissue.
  • VEGF vascular endothelial growth factor
  • FGF2 fibroblast growth factor
  • peripheral nervous system injuries are a common cause of occupational disability and such injuries not only incapacitate numerous workers or working age individuals, but reduce the quality of life. Rehabilitation of a peripheral nerve injury can require a prolonged period of treatment including periods of a year or longer. Photographs of peripheral nervous system injuries and their symptoms are shown by FIGS. 2-5 .
  • One type of the reconstructive treatment involves reconnection of the incised nerve ends by means of the end-to-end anastomosis. Peripheral nerve injuries are often accompanied by the formation of prolonged defects, thereby rendering this approach inapplicable. In such cases, autologous nerve grafting is the most appropriate option for repairing prolonged nerve defects. A nerve that is less functionally significant can be used as an autologous graft.
  • Another treatment involves replacement of a peripheral nervous system tissue defect with various structures that create conditions for peripheral nerve regeneration, such as a tubular structure that is designed to replace an extended tissue defect and foster peripheral nerve regeneration.
  • VEGF Vascular endothelial growth factor
  • VEGF is one of the well-studied growth factors that affect recovery of peripheral nerves.
  • VEGF is one of the main regulators of angiogenesis and vasculogenesis. It is a disulfide-bound dimeric glycoprotein having an average molecular weight of 34-42 kDa.
  • VEGF-A is a specific mitogen for endothelial cells (ECs) and induces their proliferation, activation, differentiation and formation of EC capillary tubules. These capillary tubules are further remodeled into mature blood vessels.
  • VEGF also induces expression of antiapoptotic proteins and increases survival of ECs. Serious defects and improper development of the cardiovascular system occurs in animals where genes encoding VEGF have been deleted. These defects may be fatal.
  • a human VEGF is encoded by a gene located on the chromosomal locus 6p21.3.
  • the coding region comprises about 14,000 bps.
  • VEGF has several isoforms including VEGF 121, VEGF 145, VEGF 148, VEGF 165, VEGF 183, VEGF 189, and VEGF 206. These isoforms result from the alternative splicing of VEGF mRNA which consists of 8 exons.
  • Different isoforms of VEGF have biochemical differences in the ability to bind heparin- and heparan-sulphate which permits them to traffic to different extracellular locations. Differences in biochemical properties or extracellular trafficking of human VEGF-A isoforms are attributable to the alternative splicing of exons 6 and 7, because all transcripts of the human VEGF-A gene contain exons 1-5 and 8.
  • VEGF had long been considered only as an inductor of angiogenesis and as a potential therapeutic agent for treatment of different disorders accompanied by tissue ischemia.
  • VEGF neuroprotective properties for neurons of both the peripheral and central nervous systems have been obtained [5, 6].
  • VEGF stimulates proliferation of Schwann cells, astrocytes, microglia, and cortical neurons [7-10].
  • a significant increase of expression of VEGF and Flt-1 (VEGF type II receptor) in the lumbar spine in response to an injury was shown in a rat sciatic nerve crush injury model [11].
  • the axonal sprouting that manifests as the increased axon number in the conduit per a unit of the cross section area was observed when VEGF was used as a part of the matrigel filling in the conduit [12].
  • VEGF-loaded poly-lactic acid microspheres in an autologous vein graft in a model of trauma with an extensive defect of fibular and tibial nerves was found to improve the nerve functional index and to increase the number of myelinated fibers in the graft [13].
  • VEGF has been shown to induce Schwann cell division and migration in a graft towards distal parts that correlates with the increased number of capillaries and myelinated fibers [14].
  • VEGF vascular endothelial growth factor
  • FGF is another growth factor that induces neurogenesis. FGF induces Schwann cell proliferation and migration in a peripheral nerve injury [16].
  • a safer method of gene transfer is based on the use of plasmid DNA.
  • intraoperative administration of a DNA plasmid comprising a vegf gene into a distal region resulted in the significantly increased number of myelinated fibers per a unit of the cross-section area of the region distal to the anastomosis site that correlated with a significant increase of the VEGF concentration in Schwann cells [21].
  • a gene-therapeutic construction could be injected paraneurally.
  • plVEGF was administrated intramuscularly and was combined with a hyaluronic acid film sheath which covered the anastomosis site in order to reduce severity of the scarring.
  • the drug intramuscular injection was accompanied by a significant increase of the muscular response amplitude and the increased number of myelinated fibers distal to the anastomosis site against their use as monotherapy [22].
  • Patent RU 2459630 C1 “Stimulation Technique for Neuroregeneration with Genetic Constructions” describes a method of the post-traumatic regeneration of the rat spinal cord when injecting a double-cassette plasmid pBud-VEGF-FGF2.
  • nucleic-acid based vectors that express growth factors such as VEGF and FGF2 and initiated studies to determine whether incorporation of these growth factors into a complex therapy of a peripheral nerve repair could be effective. As shown herein, a better, more reliable, and more effective treatment of peripheral nerve injuries is possible using a nucleic acid-based therapy.
  • one object of the present invention is to provide a method for treating a peripheral nervous system damage or injury, or for regenerating peripheral nervous system tissue, comprising administering to a subject in need thereof a vector that comprises polynucleotide sequences that encode vascular endothelia growth factor (VEGF) and fibroblast growth factor (FGF2).
  • VEGF vascular endothelia growth factor
  • FGF2 fibroblast growth factor
  • the vector comprises FGF2 encoding nucleotides at positions 699-1166 and VEGF165 encoding nucleotides at positions 3723-4298 of SEQ ID NO: 1 and resistance to kanamycin nucleotides at positions 1469-2511 of SEQ ID NO: 1.
  • the vector is pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1).
  • Another object of the present invention is to provide a vector comprising polynucleotide sequences that encode vascular endothelia growth factor (VEGF), fibroblast growth factor (FGF2), and resistance to kanamycin.
  • the vector has SEQ ID NO: 1 and comprises FGF2 encoding nucleotides at positions 699-1166, VEGF165 encoding nucleotides at positions 3723-4298 of SEQ ID NO: 1, and resistance to kanamycin nucleotides at positions 1469-2511 of SEQ ID NO: 1.
  • Another object of the present invention is to provide a cell that has been transformed with the vector.
  • FIG. 1 provides the repeated approach. The entire sciatic nerve with an autologous graft is visualized. Results after injection of plasmid pBud(Kan)-VEGF-FGF2.
  • FIG. 2 shows a view of the upper extremity prior to surgery. Post-traumatic and post-operative indented irregular scars are seen on the anterior and posterolateral surfaces of the lower, middle, and upper third of the right upper arm.
  • FIG. 3 shows lack of active movements in the middle phalanges of fingers 2-5.
  • FIG. 4 shows the impaired prehension function by all fingers.
  • FIG. 5 shows high-grade atrophy of the hand muscle within a zone innervated by the median and ulnar nerves and the ability to oppose finger 1 to finger 2 only.
  • FIG. 6 shows injection of the recombinant plasmid pBud (Kan)-VEGF-FGF2 into the repaired nerve into the suture zone and also proximally and distally over the length of 10 cm.
  • FIG. 7 shows application of fibrin glue to prevent leakage of the recombinant plasmid.
  • FIG. 8 shows atrophy of hand and forearm muscles. Nail changes: hypoplastic. Secretory function (sweating): decreased. Figure demonstrates post-surgical improvement in patient's condition.
  • FIG. 9 shows a hook grasp (a purse handle). Figure demonstrates post-surgical improvement in patient's condition.
  • FIG. 10 shows a first grasp. Figure demonstrates post-surgical improvement in patient's condition.
  • FIG. 11 shows tip prehension (finger Figure demonstrates post-surgical improvement in patient's condition.
  • FIG. 12 shows tip prehension (finger Figure demonstrates post-surgical improvement in patient's condition.
  • FIG. 13 shows tip prehension (finger I-IV). Figure demonstrates post-surgical improvement in patient's condition.
  • FIG. 14 shows a diagram of electromyography results for the thenar muscle group. Based on the electromyography results the thenar muscle response amplitude had increased over the year from 0 mV to 5 mV and almost achieved the value of the contralateral extremity.
  • FIG. 15 shows electromyography findings for the hypothenar muscle group.
  • the present invention is used in medicine, preferably in neurosurgery, traumatology and maxillofacial surgery, and in treatment of peripheral nerve injuries.
  • a goal of the inventors' research has been to create, based on their experience in the development of gene therapeutic agents, an effective product for treating patients with peripheral nerve injuries.
  • the inventors have developed various gene therapeutic constructions that differ from each other by the number of encoded transgenes and the transgenes, as well as by the nucleotide sequences of the same transgenes.
  • an object of the present invention is to provide an improved or enhanced method for reconstructive treatment involving delivery of a therapeutic polynucleotide construct into or in the vicinity of a damaged peripheral nervous system tissue.
  • An example of this embodiment is the delivery of genetic sequences encoding VEGF and FGF-2 into such tissue using the recombinant plasmid pBud(Kan)-VEGF-FGF2.
  • An object of the present invention is to provide a method for treating a peripheral nervous system damage or injury, or for regenerating peripheral nervous system tissue, comprising administering to a subject in need thereof a vector that comprises polynucleotide sequences that encode vascular endothelia growth factor (VEGF) and fibroblast growth factor (FGF2).
  • VEGF vascular endothelia growth factor
  • FGF2 fibroblast growth factor
  • a range of the injected plasmid could be from 200 to 500 ⁇ g per nerve in 2.5 ml of a physiologic saline solution.
  • the ranges include all values and subranges therebetween, including 250, 300, 350, 400, and 450 ⁇ g per nerve in 2.5 ml of a physiologic saline solution and any amount in between.
  • the vector could be administered in vivo.
  • the vector is administered to a site of the peripheral nervous system damage or injury or to a tissue to be regenerated.
  • the vector is administered to a site of the peripheral nervous system damage or injury at a site proximal or distal to the peripheral nervous system damage, or at sites proximal and distal to said damage.
  • the vector could be administered intra-, peri- and/or paraneurally.
  • the vector is contacted with a neuron or a Schwann cell, astrocyte, microglia and/or neuron.
  • the subject has neurotmesis. In another embodiment, the subject has a diastatic peripheral nerve damage. In a different embodiment, the subject has peripheral nerve damage other than neurotmesis or diastatic peripheral nerve damage.
  • the subject could be human or animal.
  • the vector comprises a polynucleotide sequence that encodes resistance to kanamycin.
  • the vector comprises FGF2 encoding nucleotides at positions 699-1166 and VEGF165 encoding nucleotides at positions 3723-4298 of SEQ ID NO: 1.
  • the vector further could comprise resistance to kanamycin nucleotides at positions 1469-2511 of SEQ ID NO: 1.
  • the vector is pBud(Kan)-VEGF-FGF2 that has SEQ ID NO: 1.
  • Another object of the present invention is to provide a vector comprising polynucleotide sequences that encode vascular endothelia growth factor (VEGF), fibroblast growth factor (FGF2), and resistance to kanamycin.
  • VEGF vascular endothelia growth factor
  • FGF2 vascular endothelia growth factor
  • FGF2 fibroblast growth factor
  • the vector comprises FGF2 encoding nucleotides at positions 699-1166 and VEGF165 encoding nucleotides at positions 3723-4298 of SEQ ID NO: 1.
  • the vector has SEQ ID NO: 1.
  • a different object of the present invention is to provide a cell that has been transformed with the vector.
  • Test animals were divided into three groups: (i) intact group, (ii) a test group where a gene therapeutic construction was administered, and (iii) a control group where a phosphate-buffered saline (PBS) solution was injected instead of the gene therapeutic construction.
  • PBS phosphate-buffered saline
  • test group (ii) a total dose of 45 ⁇ g of a gene therapeutic construction was directly injected equally into distal and proximal ends of an autologous nerve graft.
  • control group (iii) a phosphate-buffered saline (PBS) solution was injected into these locations instead of the gene therapeutic construction.
  • PBS phosphate-buffered saline
  • the evaluation criteria of the regeneration dynamics of the peripheral nerve included neurophysiological parameters such as the nerve conduction velocity and the muscle response amplitude as well as the histological examination findings such as the number of myelinated fibers and the capillary network density.
  • the neurophysiological parameters in the test group (ii) were superior to those in the control group (iii); however, they were significantly inferior to those in the intact animals of group (i).
  • the inventors have sought to determine whether the effect observed in Comparative Example 1 was attributable to the construction of the used plasmid.
  • the inventors have engineered a new plasmid encoding VEGF and FGF2 which replaced the tag sequences in the prior vector with a gene encoding kanamycin resistance.
  • plasmid pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1) was constructed.
  • This plasmid has been engineered to include a sequence encoding resistance to kanamycin at nucleotides 1469-2511 of SEQ ID NO: 1; cDNA of a gene encoding FGF2 at nucleotides 699-1166 in SEQ ID NO: 1; cDNA of the gene encoding VEGF165 at nucleotides 3723-4298 in SEQ ID NO: 1; and the Kozak sequence at nucleotides 695-698 and 3719-3722.
  • the rat animal model of a peripheral nerve injury substantially as described in Comparative Example 1 was used to evaluate the effect of administering the new plasmid constructs, including plasmid pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1).
  • Gene therapeutic constructions were administrated intraneurally immediately after the peripheral nerve suturing. The results were evaluated after 60 days following the surgical intervention and therapeutic constructs administration. Of all the plasmid DNAs that were used, the best results were obtained for the plasmid pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1) containing genetic sequences of FGF2 and VEGF.
  • FIG. 1 The results for the plasmid pBud(Kan)-VEGF-FGF2 are depicted by FIG. 1 . Based on these favorable non-clinical results, the inventors evaluated whether peripheral nerve regeneration could be attained in the clinical setting using the gene therapeutic construction pBud(Kan)-VEGF-FGF2 (SEQ ID NO: 1), as described below.
  • Hand prehension patterns the hand is unable to perform any type of prehension ( FIG. 3-4 ).
  • Diagnosis the injury of the median and ulnar nerves in the middle third of the forearm sustained 2 years ago. The status post suturing and neurolysis of the median and ulnar nerves are shown in FIG. 5 .
  • the surgery was conducted under the nerve block anaesthesia. Following triple treatment of the surgical field, an arcuate incision was made on the inner surface of the right upper arm. The median and ulnar nerves were isolated with technical difficulties. The suture lines had been found. There were no neuroma signs observed; however, the nerves were involved in a scar-forming process and adhered to the surrounding tissue.
  • the plasmid pBud(Kan)-VEGF-FGF2 was injected with an insulin needle, 250 ⁇ g per nerve in 2.5 ml of a physiologic saline solution. The injection was administered into the suture zone and also proximally and distally over the length of 10 cm ( FIG. 6 ). After that 2 ml of the two-component fibrin glue TISSUCOL® was applied to the isolated nerves ( FIG. 7 ).
  • the post-surgical case included hemostasis, wound suturing, placement of a rubber tube drainage, and application of an antiseptic dressing and a plaster cast. A re-examination was performed one month after the surgery.
  • Hand prehension patterns the hand is unable to perform any type of prehension.

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RU2014137218/10A RU2558294C1 (ru) 2014-09-16 2014-09-16 Кодон-оптимизированная рекомбинантная плазмида, способ стимуляции регенерации периферического нерва, способ лечения поврежденного нерва человека
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EP3196307B1 (en) 2018-12-26
JP2017532021A (ja) 2017-11-02
US20170319658A1 (en) 2017-11-09
CN107087417A (zh) 2017-08-22
AU2015390821A1 (en) 2017-05-04
AU2015390821B2 (en) 2018-08-09
WO2016163912A1 (ru) 2016-10-13
JP6540797B2 (ja) 2019-07-10
RU2558294C1 (ru) 2015-07-27
US10434145B2 (en) 2019-10-08
BR112017005310A2 (pt) 2018-02-14
BR112017005310B1 (pt) 2023-10-03

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