Classifications

• • 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
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present invention relates to gene therapy. More particularly, the invention relates to gene therapy compositions and methods of using the same to treat neuronal ceroid lipofuscinoses.
• NCLs Neuronal ceroid lipofuscinoses
• NCLs Neuronal ceroid lipofuscinoses
• Most NCLs are autosomal recessive diseases. NCLs typically begin in early childhood and affect an estimated 2 to 4 of every 100,000 individuals. These disorders appear to be more common in Finland, Sweden, other parts of northern Europe, and Newfoundland, Canada. Early symptoms of these disorders include subtle personality and behavior changes, slow learning, clumsiness, or stumbling. As the disease progresses, affected children suffer cognitive impairment, worsening seizures, and progressive loss of sight and motor skills. Eventually, children with NCL become blind, bedridden, and demented.
• Batten disease (Shmeyer-Vogt-Sjogren-Batten disease) is the most common NCL. Although Batten disease originally referred specifically to the juvenile form of NCL (JNCL), the term Batten disease is increasingly used by pediatricians to describe all forms of NCL. There are four other main types of NCL, including three forms that begin earlier in childhood and a very rare form that strikes adults. The symptoms of these childhood types are similar to those caused by Batten disease, but they become apparent at different ages and progress at different rates.
• Congenital NCL is a very rare and severe form of NCL; babies have microcephaly and seizures, and die soon after birth.
• Infantile NCL (INCL or Santavuori-Haltia disease) begins between about ages 6 months and 2 years and is marked by failure to thrive, microcephaly, and myoclonic contractions. These children usually die before age 5.
• Late infantile NCL (LINCL, or Jansky-Bielschowsky disease) begins between ages 2 and 4 and results in loss of muscle coordination (ataxia), seizures, and eventually death between ages 8 and 12.
• Adult NCL (Kufs disease, Parry's disease, and ANCL) usually manifests before age 40, causes milder symptoms that progress slowly, and does not cause blindness.
• NCLs While enzyme replacement therapies have recently provided somewhat encouraging results in improving the neurological manifestations of some forms of Batten disease, they do not address the full spectrum of Batten disease manifestations, and their administration is associated with significant inconvenience. Stem cell therapies have yielded disappointing results. Most available treatments, only treat the symptoms of NCLs: seizures may be controlled with anticonvulsant drugs, physical and occupational therapy may help patients retain function as long as possible, and support and encouragement can help patients and families cope with the profound disability and dementia caused by NCLs.
• the invention generally relates, in part, to gene therapy compositions and methods for the treatment, prevention, or amelioration of at least one symptom of neuronal ceroid lipofuscinoses.
• a polynucleotide comprising: a left (5 ' ) lentiviral LTR; a Psi ( ⁇ ) packaging signal; a retroviral export element; a central polypurine tract DNA flap (cPPT/FLAP); a promoter operably linked to a polynucleotide encoding a tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral LTR.
• the lentivirus is selected from the group consisting of: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
• HIV human immunodeficiency virus
• VMV visna-maedi virus
• CAEV caprine arthritis-encephalitis virus
• EIAV equine infectious anemia virus
• FV feline immunodeficiency virus
• BIV bovine immune deficiency virus
• SIV simian immunodeficiency virus
• the lentivirus is HIV-1 or HIV -2.
• the lentivirus is HIV- 1.
• the promoter of the 5 ' LTR is replaced with a heterologous promoter selected from the group consisting of: a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, and a Simian Virus 40 (SV40) promoter.
• CMV cytomegalovirus
• RSV Rous Sarcoma Virus
• SV40 Simian Virus 40
• the 3 ' LTR comprises one or more modifications.
• the 3 ' LTR comprises one or more deletions that prevent viral transcription beyond the first round of viral replication.
• the 3 ' LTR comprises a deletion of the TATA box and Spl and NF- ⁇ transcription factor binding sites in the U3 region of the 3 ' LTR.
• the 3' LTR is a self-inactivating (SIN) LTR.
• the promoter operably linked to a polynucleotide encoding a TPP1 polypeptide comprises a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter or transcriptionally active fragment thereof.
• MND primer-binding site substituted
• the promoter operably linked to a polynucleotide encoding a TPP1 polypeptide is selected from the group consisting of: integrin subunit alpha M (ITGAM; CDl lb), CD68, C-X3-C motif chemokine receptor 1 (CX3CR1), ionized calcium binding adaptor molecule 1 (IBA1), transmembrane protein 119 (TMEM119), spalt like transcription factor 1 (SALL1) and adhesion G protein-coupled receptor El (F4/80).
• the promoter operably linked to a polynucleotide encoding a TPPl polypeptide comprises an elongation factor 1 alpha (EF1 ⁇ ) promoter or transcriptionally active fragment thereof.
• EF1 ⁇ elongation factor 1 alpha
• the promoter operably linked to a polynucleotide encoding a TPPl polypeptide is a short EF1 ⁇ promoter.
• the promoter operably linked to a polynucleotide encoding a TPPl polypeptide is a long EF1 ⁇ promoter.
• the polynucleotide encoding the TPPl polypeptide is a cDNA.
• the polynucleotide encoding the TPPl polypeptide is codon optimized for expression.
• a polynucleotide comprising: a left (5 ' ) HIV-1 LTR; a Psi ( ⁇ ) packaging signal; an RRE retroviral export element; a cPPT/FLAP; an MND promoter or EF1 ⁇ promoter operably linked to a polynucleotide encoding a TPPl polypeptide; and a right (3 ) HrV-1 LTR.
• a polynucleotide comprising: a left (5 ' ) CMV promoter/HIV- 1 chimeric LTR; a Psi ( ⁇ ) packaging signal; an RRE retroviral export element; a cPPT/FLAP; an MND promoter or EF1 ⁇ promoter operably linked to a polynucleotide encoding a TPPl polypeptide; and a right (3 ' ) SIN HIV-1 LTR.
• the polynucleotide further comprise a bovine growth hormone polyadenylation signal or a rabbit ⁇ -globin polyadenylation signal.
• a mammalian cell transduced with a lentiviral vector comprising a polynucleotide contemplated herein.
• the cell is a hematopoietic cell.
• the cell is a CD34+ cell.
• the cell is a stem cell or progenitor cell.
• a producer cell comprising: a first polynucleotide encoding gag, a second polynucleotide encoding pol, a third polynucleotide encoding env, and a polynucleotide contemplated herein.
• a lentiviral vector produced by the producer cell contemplated herein is provided.
• a composition comprising a lentiviral vector comprising a polynucleotide or a mammalian cell contemplated herein is provided.
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lentiviral vector comprising a polynucleotide or a mammalian cell contemplated herein is provided.
• a method of treating neuronal ceroid lipofuscinoses comprising administering to a subject a lentiviral vector comprising a polynucleotide, a cell transduced with a lentiviral vector comprising a polynucleotide, or a mammalian cell contemplated herein is provided.
• NCL neuronal ceroid lipofuscinoses
• a method of treating neuronal ceroid lipofuscinoses comprising administering to a subject a pharmaceutical composition contemplated herein is provided.
• a method of decreasing at least one symptom associated with neuronal ceroid lipofuscinoses in a subject comprising administering to a subject a lentiviral vector comprising a polynucleotide, a cell transduced with a lentiviral vector comprising a polynucleotide, or a mammalian cell contemplated herein is provided.
• a method of decreasing at least one symptom associated with neuronal ceroid lipofuscinoses in a subject comprising administering to a subject a pharmaceutical composition contemplated herein.
• At least one symptom is selected from the group consisting of: seizures, loss of vision, cognitive function decline, and motor function decline.
• the subject has been diagnosed with late-infantile NCL (LINCL).
• LINCL late-infantile NCL
• the subject has been diagnosed with juvenile Batten Disease (JNCL).
• JNCL juvenile Batten Disease
• Figure 1 shows exemplary architectures of lentiviral vectors encoding TPP1.
• FIG. 2 shows a representative Western blot of TPP1 expression in wild type fibroblasts, TPPl -/- fibroblasts, and TPPl -/- fibroblasts transduced with pMND-TPPl or pEFl ⁇ -TPPl.
• the lower arrow indicates the ⁇ -actin control protein at its expected size of ⁇ 42kDa in all samples.
• TPP-1 protein (upper arrow) was detected at low levels in the wild type fibroblasts and was overexpressed in transduced ⁇ 1 -/- fibroblasts (right-most two lanes).
• Figure 3A shows the data from a representative experiment assaying TPP1 enzymatic activity in wild type control cells, ⁇ 1 -/- cells, and ⁇ 1 -/- cells transduced with the lentiviral vectors encoding TPPl (pMND-TPPl and pEFl ⁇ -TPPl).
• Figure 3B shows that ⁇ 1 -/- fibroblasts transduced with lentiviral vectors encoding TPPl secrete about 10-fold more active TPPl into cell culture supernatant compared to background levels assayed in wild type cells and untransduced ⁇ 1 -/- fibroblasts.
• FIG. 4 shows that F108 and PGE2 increase transduction with lentiviral vectors encoding tripeptidyl peptidase 1 (TPPl).
• Human CD34 + cells were transduced with LVVs encoding either EFl ⁇ -TPPl or MND-TPP1 at an MOI of 5 or 15 with and without F108 and PGE2.
• Day 14 pooled methylcellulose colony VCNs are shown in upper left panel.
• TPP-1 enzyme activity from day 7 cytokine cultures are shown in upper middle panel (cell pellet) and upper right panel (cell supernatant).
• Lineage negative (Lin-) bone marrow cells were transduced with an MND-TPPl LVV at an MOI of 5, 15, or 30 with and without F108 and PGE2.
• D7 liquid culture VCNs are shown in the lower left panel; individual colony VCN by qPCR is shown in the lower middle panel; and %LVV+ colonies are shown in the lower right panel.
• Figure 5 shows that hCD34 + cell growth was not adversely affected by transducing the cells with lentiviral vector in the presence of F 108 and PGE2.
• Figure 6 shows that hCD34 + cells transduced with a pMND-CLN2 LVV or pEFl ⁇ CLN2 LVV in the presence of F 108 and PGE2 and cultured for 14 days had increased VCNs across all MOIs for both lentiviral vectors.
• Figure 7 shows that hCD34 + cells transduced with a pMND-CLN2 LVV or pEFl ⁇ CLN2 LVV in the presence of F 108 and PGE2 and cultured for 14 days in methylcellulose had increased VCNs and %LVV+ cells when measured from individual colonies (left panel). Transduction did not significantly affect colony formation (right panel).
• FIG 8 shows that hCD34 + cells transduced with a pMND-CLN2 LVV or pEFl ⁇ CLN2 LVV in the presence of F 108 and PGE2 show increased TPP-1 activity.
• Figure 9 shows confocal microscopy for TPPl (to detect TPPl expression) and ATP synthase subunit c (to detect TPPl activity) in untranduced wild type induced pluripotent stem cells (iPSCs), untransduced patient-derived TPPl -/- iPSCs, or patient- derived TPP -/- iPSCs transduced with lentiviral vectors (LVVs) comprising an MND or EF l ⁇ -short promoter operably linked to a polynucleotide encoding TPPl.
• iPSCs wild type induced pluripotent stem cells
• LVVs lentiviral vectors
• TPPl expression is present and subunit c expression is absent in
• SEQ ID NO: 1 sets forth the sequence of an exemplary lentiviral vector encoding TTP 1.
• SEQ ID NO: 2 sets forth the sequence of an exemplary lentiviral vector encoding TTP 1.
• SEQ ID NO: 3 sets forth a polynucleotide sequence encoding a human tripeptidyl peptidase 1 (TPPl) polypeptide.
• SEQ ID NO: 4 sets forth a codon-optimized polynucleotide sequence encoding a human tripeptidyl peptidase 1 (TPPl) polypeptide.
• SEQ ID NO: 5 sets forth an amino acid sequence encoding a human tripeptidyl peptidase 1 (TPPl) polypeptide.
• SEQ ID NOs: 6-16 set forth the amino acid sequences of various linkers.
• SEQ ID NOs: 17-19 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.
• the invention generally relates, in part, to improved gene therapy compositions and methods for treating, preventing, or ameliorating at least one symptom of a neuronal ceroid lipofuscinoses (NCL).
• NCL neuronal ceroid lipofuscinoses
• NCLs are a group of inherited disorders of the nervous system for which there is no clinically approved curative treatment and for which palliative care is the only option.
• NCLs are lysosomal storage diseases, marked by a buildup of substances called lipofuscins or lipopigments in membrane bound organelles called lysosomes. Lysosomes are compartments in the cell that normally digest and recycle different types of molecules. The accumulation of lipofuscins in lysosomes occurs in cells throughout the body, but neurons in the brain seem to be particularly vulnerable to the damage caused by lipofuscins. The progressive death of cells, especially in the central nervous system, leads to vision loss, seizures, and decline in motor function and cognitive ability in people with NCLs.
• Late-infantile NCL (LINCL or CLN2) is a devastating childhood neuronal ceroid lipofuscinosis disease caused by deficiency in tripeptidyl peptidase 1 (TTP1).
• TTP1 tripeptidyl peptidase 1
• At least 100 mutations in the TPPl gene have been found to cause LINCL.
• Most mutations in the TPPl gene alter single amino acids in tripeptidyl peptidase 1 , resulting in a severe decrease in enzymatic activity.
• the mutations IV5-1G>C and R508X cause nearly 90 percent of cases of LINCL worldwide. Loss of TPPl function leads to accumulation of lipofuscin deposits in the lysosomes causing cell damage and atrophy with cells of the nervous system most acutely affected.
• LINCL patients first experience symptoms between the ages of 2 and 4 years old, progressively loosing neurological function beginning with blindness, ataxia, dementia, and then death, usually before the second decade of life (Anderson et al, 2013. Biochimica et Biophysica Acta. 1832: 1807-1826).
• JNCL juvenile Batten disease
• Children with JNCL develop progressive vision loss, intellectual and motor disability, speech difficulties, and seizures. Vision impairment is often the first noticeable sign of JNCL, beginning between the ages of 4 and 8 years. Vision loss tends to progress rapidly, eventually resulting in blindness. After vision impairment has begun, children with juvenile Batten disease experience the loss of previously acquired skills (developmental regression), usually beginning with the ability to speak in complete sentences. Affected children also have difficulty learning new information. In addition to the intellectual decline, affected children lose motor skills such as the ability to walk or sit. They also develop movement abnormalities that include rigidity or stiffness, slow or diminished movements (hypokinesia), and stooped posture. Affected children may have recurrent seizures (epilepsy), heart problems, behavioral problems, difficulty sleeping, and problems with attention that begin in mid- to late childhood. Most people with JNCL live into their twenties or thirties.
• a gene therapy vector encoding a tripeptidyl peptidase 1 (TPPl) polypeptide is contemplated.
• the gene therapy preferentially includes a promoter operably linked to the polynucleotide encoding the TPP1 polypeptide.
• the gene therapy vector may be a viral vector, including but not limited to a gammaretroviral vector, a lentiviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, or a herpes virus vector.
• the transduced cells are hematopoietic cells, including, but not limited to CD34 + cells.
• gene therapy compositions contemplated herein are preferably administered to a subject that has a neuronal ceroid lipofuscinosis, more preferably a subject that has late-infantile NCL (LINCL) or juvenile Batten Disease (JNCL) or even more preferably, a subject that has one or more mutations in a TPP1 gene.
• LINCL late-infantile NCL
• JNCL juvenile Batten Disease
• an element means one element or one or more elements.
• the term "about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• the term "about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
• the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
• the term “substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• hematopoietic stem or progenitor cell transduced with compositions and methods contemplated herein comprises an increase in the number of transduced cells compared to existing transduction compositions and methods. Increases in cell transduction, can be ascertained using methods known in the art, such as reporter assays, RT-PCR, and cell surface protein expression, among others.
• An “increased” or “enhanced” amount of transduction is typically a "statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the number of cells transduced by vehicle, a control composition, or other transduction method.
• a “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to compositions or methods that result in comparably fewer transduced cells compared to cells transduced with compositions and/or methods according to the present invention.
• a “decrease” or “reduced” amount of transduced cells is typically a "statistically significant” amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the number of transduced cells (reference response) produced by compositions and/or methods according to the present invention.
• maintain or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, a control molecule/composition, or the response in a particular cell.
• a comparable response is one that is not significantly different or measurable different from the reference response.
• Polypeptide “polypeptide fragment,” “peptide” and “protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.
• a “polypeptide” includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques.
• Polypeptides are not limited to a specific length, e.g., they may comprise a full length protein sequence, a fragment of a full length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
• polypeptides are contemplated herein, including, but not limited to, TPP1 polypeptides, e.g., SEQ ID NO: 5.
• isolated peptide or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
• Polypeptides include "polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to modulate the biological properties of a polypeptide by introducing one or more substitutions, deletions, additions and/or insertions into the polypeptide.
• polypeptides include polypeptide variants having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence.
• Polypeptides variants include biologically active "polypeptide fragments.”
• biologically active fragment or “minimal biologically active fragment” refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity.
• Polypeptide fragments refer to a polypeptide, which can be monomeric or multimeric that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion or substitution of one or more amino acids of a naturally -occurring or recombinantly -produced polypeptide.
• a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long.
• polypeptide fragments include catalytic domains and the like.
• polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al , (1987 , Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D.
• a variant will contain one or more conservative substitutions.
• a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments, polypeptides include polypeptides having at least about and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant polypeptide, one skilled in the art, for example, can change one or more of the codons of the encoding DNA sequence.
• amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i. e., substitutions of similarly charged or uncharged amino acids.
• a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
• Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
• hydropathic index of amino acids may be considered.
• the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
• hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
• methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
• amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
• substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
• amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
• Polypeptide variants further include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules).
• Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art.
• Variants also include allelic variants, species variants, and muteins.
• Fusion polypeptides contemplated in particular embodiments include fusion polypeptides.
• fusion polypeptides and polynucleotides encoding fusion polypeptides are provided.
• Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
• two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.
• Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, cell permeable peptide domains (CPP), DNA binding domains, nuclease domains, chromatin remodeling domains, histone modifying domains, epigenetic modifying domains, exodomains, extracellular ligand binding domains, antigen binding domains, transmembrane domains, intracellular signaling domains, multimerization domains, epitope tags (e.g., maltose binding protein ("MBP"), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals.
• MBP maltose binding protein
• GST glutathione S transferase
• Fusion polypeptides are typically linked C- terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N- terminus to N-terminus, or N-terminus to C-terminus.
• the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques. Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.
• Fusion polypeptides may optionally comprises a linker that can be used to link the one or more polypeptides or domains within a polypeptide.
• a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions
• Exemplary linkers include, but are not limited to the following amino acid sequences: glycine polymers (G)n; glycine-serine polymers (Gl-5Sl-5)n, where n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; GGG (SEQ ID NO: 6); DGGGS (SEQ ID NO: 7); TGEKP (SEQ ID NO: 8) (see e.g., Liu et ctl, PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 9) (Pomerantz et al.
• LRQRDGERP SEQ ID NO: 14
• LRQKDGGGSERP SEQ ID NO: 15
• LRQKd(GGGS)2ERP SEQ ID NO: 16
• flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993)) or by phage display methods.
• Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template.
• a polypeptide cleavage site can be put into any linker peptide sequence.
• Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
• Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al, 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).
• Exemplary protease cleavage sites include, but are not limited to the cleavage sites of poty virus NIa proteases (e.g., tobacco etch virus protease), poty virus HC proteases, potyvirus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2- encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
• poty virus NIa proteases e.g., tobacco etch virus protease
• poty virus HC proteases e.g
• TEV tobacco etch virus protease cleavage sites
• EXXYXQ(G/S) SEQ ID NO: 17
• ENLYFQG SEQ ID NO: 18
• ENLYFQS SEQ ID NO: 19
• the self-cleaving polypeptide site comprises a 2A or 2A- like site, sequence or domain (Donnelly et al, 2001. J. Gen. Virol. 82: 1027-1041).
• the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
• polypeptides or fusion polypeptides contemplated herein is regulated by one or more protein destabilization sequences or protein degradation sequences (degrons).
• destabilization sequences or protein degradation sequences degrons.
• Illustrative examples of protein destabilization sequences include, but are not limited to: the destabilization box (D box), a nine amino acid is present in cell cycle- dependent proteins that must undergo rapid and complete ubiquitin-mediated proteolysis to achieve cycling within the cell cycle (see e.g., Yamano et al. 1998. Embo J 17 :5670-8); the KEN box, an APC recognition signal targeted by Cdhl (see e.g., Vietnameser et al. 2000.
• D box destabilization box
• APC recognition signal targeted by Cdhl see e.g., Pfleger et al. 2000.
• degrons suitable for use in particular embodiments include, but are not limited to, ligand controllable degrons and temperature regulatable degrons.
• ligand controllable degrons include those stabilized by Shield 1 (see e.g., Bonger et al. 2011. Nat Chem Viol. 7(8):531-537), destabilized by auxin (see e.g., Nishimura et al. 2009. Nat Methods 6(12):917-922), and stabilized by
• trimethoprim see e.g., Iwamoto et al, 2010. Chem Biol. 17(9):981-8).
• Non-limiting examples of temperature regulatable degrons include, but are not limited to DHFRTS degrons (see e.g., Dohmen et al, 1994. Science 263(5151): 1273-1276).
• a polypeptide contemplated herein comprises one or more degradation sequences selected from the group consisting of: a D box, an O box, an A box, a KEN motif, a PEST motifs, Cyclin A and UFD domain/substrates, ligand controllable degrons, and temperature regulatable degrons.
• polynucleotide or “nucleic acid” refer to
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• DNA/RNA hybrids DNA/RNA hybrids
• Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated.
• Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
• pre-mRNA pre-messenger RNA
• mRNA messenger RNA
• RNA short interfering RNA
• shRNA short hairpin RNA
• miRNA microRNA
• ribozymes synthetic RNA
• genomic RNA gRNA
• Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths. It will be readily understood that "intermediate lengths," in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.
• polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
• polynucleotides may be codon-optimized.
• codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
• Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, and/or (x) systematic variation of codon sets for each amino acid.
• polynucleotides include, but are not limited to
• polynucleotides contemplated herein include, but are not limited to polynucleotides comprising expression vectors, viral vectors, transfer plasmids, expression cassettes and polynucleotides encoding a tripeptidyl peptidase 1 (TPP1) polypeptide.
• TPP1 tripeptidyl peptidase 1
• TPP1 The tripeptidyl peptidase 1 (TPP1) gene encodes TPP1 (also referred to as CLN2,
• TPP1 encodes a 563-amino acid preproenzyme with a 19-amino acid signal peptide and a 176-amino acid prodomain that are removed during maturation, yielding a 368-amino acid mature enzyme. TPP1 also contains 5 N-glycosylation sites.
• the proenzyme has an apparent molecular mass of 66 kD, and the mature enzyme has an apparent molecular mass of 46 to 48 kD.
• TPP1 is a lysosomal exopeptidase that sequentially cleaves N-terminal tripeptides from substrates, and has weaker endopeptidase activity.
• TPP1 is synthesized as a catalytically -inactive enzyme which is activated and auto-proteolyzed upon acidification. Mutations in the TPP1 gene result in late-infantile neuronal ceroid lipofuscinosis and a rare form of juvenile Batten disease, which are associated with the failure to degrade specific neuropeptides and a subunit of ATP synthase in the lysosome.
• polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
• sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide- by -nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
• a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g.
• A, T, C, G, I or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met
• nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
• isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state, e.g. , a DNA fragment that has been removed from the sequences that are normally adj acent to the fragment.
• an isolated polynucleotide refers to a
• cDNA complementary DNA
• a recombinant polynucleotide a recombinant polynucleotide, a synthetic polynucleotide, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
• polynucleotides include: 5' (normally the end of the polynucleotide having a free phosphate group) and 3' (normally the end of the polynucleotide having a free hydroxyl (OH) group).
• Polynucleotide sequences can be annotated in the 5' to 3' orientation or the 3' to 5' orientation.
• the 5' to 3' strand is designated the "sense,” "plus,” or "coding” strand because its sequence is identical to the sequence of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA].
• the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as "template,” "antisense,” “minus,” or “non-coding” strand.
• reverse orientation refers to a 5' to 3' sequence written in the 3' to 5' orientation or a 3' to 5' sequence written in the 5' to 3' orientation.
• complementarity refers to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the
• complementary strand of the DNA sequence 5 ⁇ G T C A T G 3' is 3' T C A G T A C 5'.
• the latter sequence is often written as the reverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'.
• a sequence that is equal to its reverse complement is said to be a palindromic sequence.
• Complementarity can be "partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there can be “complete” or “total” complementarity between the nucleic acids.
• nucleic acid cassette refers to genetic sequences within the vector which can express an RNA, and subsequently a polypeptide.
• the nucleic acid cassette contains a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest.
• the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest.
• Vectors may comprise one, two, three, four, five or more nucleic acid cassettes.
• the nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments.
• the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.
• the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder.
• the cassette can be removed and inserted into a plasmid or viral vector as a single unit.
• polynucleotide(s)-of-interest refers to one or more polynucleotides, e.g., a polynucleotide encoding a polypeptide (i.e., a polypeptide-of- interest), inserted into an expression vector that is desired to be expressed.
• vectors and/or plasmids of the present invention comprise one or more polynucleotides-of-interest, e.g., a polynucleotide encoding a TPP1 polypeptide.
• a polynucleotide-of-interest encodes a polypeptide that provides a therapeutic effect in the treatment, prevention, or amelioration of a neuronal ceroid lipofuscinoses, which may be referred to as a "therapeutic polypeptide," e.g. , a
• a polynucleotide-of-interest comprises an inhibitory polynucleotide including, but not limited to, a crRNA, a tracrRNA, a single guide RNA (sgRNA), an siRNA, an miRNA, an shRNA, a ribozyme or another inhibitory RNA.
• an inhibitory polynucleotide including, but not limited to, a crRNA, a tracrRNA, a single guide RNA (sgRNA), an siRNA, an miRNA, an shRNA, a ribozyme or another inhibitory RNA.
• Polynucleotides regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, post-transcription response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a
• polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
• Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
• a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
• vectors include, but are not limited to plasmid,
• vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
• artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC)
• bacteriophages such as lambda phage or Ml 3 phage
• animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
• viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
• retrovirus including lentivirus
• adenovirus e.g., adeno-associated virus
• herpesvirus e.g., herpes simplex virus
• poxvirus baculovirus
• papillomavirus papillomavirus
• papovavirus e.g., SV40
• expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V 5-DESTTM, pLenti6/V 5- DESTTM, and pLenti6.2/V 5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
• coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
• the vector is an episomal vector or a vector that is maintained extrachromosomally.
• episomal vector refers to a vector that is able to replicate without integration into host's chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates
• “Expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector— origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, post-transcriptional regulatory elements, a polyadenylation sequence, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used.
• a polynucleotide is a vector, including but not limited to expression vectors and viral vectors, and includes exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers.
• An "endogenous” control sequence is one which is naturally linked to a given gene in the genome.
• An “exogenous” control sequence is one which is placed in juxtaposition to a gene by means of genetic
• a "heterologous" control sequence is an exogenous sequence that is from a different species than the cell being genetically manipulated.
• a “synthetic" control sequence may comprise elements of one more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in silico that provide optimal promoter and/or enhancer activity for the particular gene therapy.
• promoter refers to a recognition site of a polynucleotide
• promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
• enhancer refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
• An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
• promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
• operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence.
• a constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a "cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
• Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g. , early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and PI 1 promoters from vaccinia virus, a short elongation factor 1 -alpha (EF1 ⁇ -short) promoter, a long elongation factor 1 -alpha (EF la-long) promoter, early growth response 1 (EGRl), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4
• CAG cytomegalovirus enhancer/chicken ⁇ -actin
• MND myeloproliferative sarcoma virus enhancer
• a cell, cell type, cell lineage or tissue specific expression control sequence may be desirable to use to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
• tissue specific promoters include, but are not limited to: an B29 promoter (B cell expression), a runt transcription factor (CBFa2) promoter (stem cell specific expression), an CD 14 promoter (monocytic cell expression), an CD43 promoter (leukocyte and platelet expression), an CD45 promoter (hematopoietic cell expression), an CD68 promoter (macrophage expression), a CYP450 3A4 promoter (hepatocyte expression), an desmin promoter (muscle expression), an elastase 1 promoter (pancreatic acinar cell expression, an endoglin promoter (endothelial cell expression), a fibroblast specific protein 1 promoter (FSP1) promoter (fibroblast cell expression), a fibronectin promoter (fibroblast cell expression), a fms-related tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), a glial fibrillary acidic protein (GFAP) promote
• expression control sequences suitable for use in particular embodiments contemplated herein include, but are not limited to expression control sequences isolated from: integrin subunit alpha M (ITGAM; CD1 lb), CD68, C- X3-C motif chemokine receptor 1 (CX3CR1), ionized calcium binding adaptor molecule 1 (IB Al ), transmembrane protein 119 (TMEM 119), spalt like transcription factor 1 (S ALL 1 ) and adhesion G protein-coupled receptor El (F4/80).
• IGAM integrin subunit alpha M
• CD1 lb CD68
• CX3-C motif chemokine receptor 1 CX3CR1
• IB Al ionized calcium binding adaptor molecule 1
• TMEM 119 transmembrane protein 119
• S ALL 1 spalt like transcription factor 1
• adhesion G protein-coupled receptor El F4/80
• condition expression may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression.
• Certain embodiments provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.
• inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone),
• metallothionine promoter inducible by treatment with various heavy metals
• MX-1 promoter inducible by interferon
• the "GeneSwitch” mifepristone-regulatable system Sirin et al, 2003, Gene, 323:67
• the cumate inducible gene switch WO 2002/088346
• tetracycline-dependent regulatory systems etc.
• polynucleotides comprises at least one (typically two) site(s) for recombination mediated by a site specific recombinase.
• recombinase or "site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten or more.), which may be wild-type proteins ⁇ see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof.
• Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ⁇ DC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.
• the polynucleotides may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector.
• recombination sequence refers to a particular nucleic acid sequence to which a recombinase recognizes and binds.
• loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)).
• exemplary loxP sites include, but are not limited to: lox511 (Hoess e/ a/., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al, 2002), lox71 (Albert et al, 1995), and lox66 (Albert et al, 1995).
• Suitable recognition sites for the FLP recombinase include, but are not limited to: FRT (McLeod, et al, 1996), Fl, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al, 1988), FRT(RE) (Senecoff et al, 1988).
• recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme ⁇ Integrase, e.g., phi-c31.
• the ⁇ C 31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al. , 2000).
• attB and attP named for the attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by ⁇ C 31 homodimers (Groth et al. , 2000).
• the product sites, attL and attR, are effectively inert to further ⁇ C31 - mediated recombination (Belteki et al. , 2003), making the reaction irreversible.
• attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagarajan et al. , 2001 ; Belteki et al, 2003).
• typical strategies position by homologous recombination an attP- bearing "docking site" into a defined locus, which is then partnered with an attB-bearing incoming sequence for insertion.
• the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.
• an "internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al, 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
• IRES immunoglobulin heavy-chain binding protein
• VEGF vascular endothelial growth factor
• FGF-2 fibroblast growth factor 2
• IGFII insulin-like growth factor
• eIF4G translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4
• EMCV encephelomy carditis virus
• IRES have also been reported in viral genomes of Picomaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
• the IRES used in polynucleotides contemplated herein is an EMCV IRES.
• the polynucleotides comprise polynucleotides that have a consensus Kozak sequence and that encode a desired polypeptide.
• the term "Kozak sequence” refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation.
• the consensus Kozak sequence is (GCC)RCCATGG [SEQ ID NO:20], where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res.
• heterologous nucleic acid transcripts increases heterologous gene expression.
• Transcription termination signals are generally found downstream of the polyadenylation signal.
• vectors comprise a polyadenylation sequence 3' of a
• polyA site or "polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
• Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a polyA tail are unstable and are rapidly degraded.
• Illustrative examples of polyA signals that can be used in a vector includes an ideal polyA sequence (e.g., AATAAA, ATT AAA,
• BGHpA bovine growth hormone polyA sequence
• r ⁇ gpA rabbit ⁇ -globin polyA sequence
• AGTAAA AGTAAA
• BGHpA bovine growth hormone polyA sequence
• r ⁇ gpA rabbit ⁇ -globin polyA sequence
• a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation.
• the suicide gene is not immunogenic to the host harboring the polynucleotide or cell.
• a certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
• polynucleotides comprise gene segments that cause the genetically modified cells contemplated herein to be susceptible to negative selection in vivo.
• Negative selection refers to an infused cell that can be eliminated as a result of a change in the in vivo condition of the individual.
• the negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound.
• Negative selection genes include, but are not limited to: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase.
• HSV-I TK Herpes simplex virus type I thymidine kinase
• HPRT hypoxanthine phosphribosyltransferase
• APRT cellular adenine phosphoribosyltransferase
• genetically modified cells comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro.
• the positive selectable marker may be a gene, which upon being introduced into the host cell, expresses a dominant phenotype permitting positive selection of cells carrying the gene.
• Genes of this type are known in the art, and include, but are not limited to hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.
• hph hygromycin-B phosphotransferase gene
• DHFR dihydrofolate reductase
• ADA adenosine deaminase gene
• MDR multi-drug resistance
• the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker.
• the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other.
• An example of a fused polynucleotide that yields as an expression product a polypeptide that confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. See also the publications of PCT US91/08442 and PCT/US94/05601, by S. D. Lupton, describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable markers with negative selectable markers.
• Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt
• preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.
• Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include, but are not limited to genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker.
• vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
• the transferred nucleic acid is generally linked to, e.g. , inserted into, the vector nucleic acid molecule.
• a vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
• Illustrative examples of vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
• Illustrative methods of delivering polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, poly cation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE- dextran-mediated transfer, gene gun, and heat-shock.
• polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosy stems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
• Lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10: 180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011 : 1 - 12.
• Antibody -targeted, bacterially derived, non-living nanocell-based delivery is also contemplated in particular embodiments.
• polynucleotides encoding one or more therapeutic polypeptides, or fusion polypeptides may be introduced into a target cell by viral methods.
• Polynucleotides encoding one or more therapeutic polypeptides, or fusion polypeptides may be introduced into a target cell by non-viral or viral methods.
• polynucleotides encoding a TPP1 polypeptide are introduced into a target cell using a vector, preferably a viral vector, more preferably a retroviral vector, and even more preferably, a lenti viral vector.
• viral vector is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a virus or viral particle that mediates nucleic acid transfer.
• Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).
• viral vector systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.
• AAV adeno-associated virus
• retrovirus retrovirus
• herpes simplex virus adenovirus
• vaccinia virus vectors for gene transfer include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.
• Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455- 460).
• the term "retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a "pro virus.”
• the pro virus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.
• Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.
• M-MuLV Moloney murine leukemia virus
• MoMSV Moloney murine sarcoma virus
• HaMuSV Harvey murine sarcoma virus
• MuMTV murine mammary tumor virus
• GaLV gibbon ape leukemia virus
• FLV feline leukemia virus
• RSV Rous Sarcoma Virus
• lentivirus refers to a group (or genus) of complex retroviruses.
• Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HrV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus
• HIV based vector backbones i.e., HIV cis-acting sequence elements
• a lentivirus is used to deliver a polynucleotide encoding a TPP1 polypeptide to a cell.
• viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself.
• Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus.
• the term "retroviral vector” refers to a viral vector or plasniid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.
• lentiviral vector refers to a viral vector or plasniid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.
• hybrid vector refers to a vector, LTR or other nucleic acid containing both retroviral, e.g., lentiviral, sequences and non-lenti viral viral sequences.
• a hybrid vector refers to a vector or transfer plasniid comprising retroviral e.g., lentiviral, sequences for reverse transcription, replication, integration and/or packaging.
• lentiviral vector may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles.
• elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements are present in RNA form in the lentiviral particles and are present in DNA form in the DNA plasmids.
• a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT/FLAP, a Psi ( ⁇ ) packaging signal, an export element, a promoter operably linked to a polynucleotide encoding a TPP1 polypeptide, a poly (A) sequence, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
• accessory elements a cPPT/FLAP, a Psi ( ⁇ ) packaging signal, an export element, a promoter operably linked to a polynucleotide encoding a TPP1 polypeptide, a poly (A) sequence, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
• lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus.
• integration defective lentivirus or “refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration- incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety.
• Illustrative mutations in the HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51 A, Q53C, D55V, D64E, D64V, E69A, K71 A, E85A, E87A, Dl 16N, Dl 161, Dl 16A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W
• LTR long terminal repeat
• the term "long terminal repeat (LTR)" refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions.
• the LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site).
• the term "packaging signal” or “packaging sequence,” “psi” and the symbol “ ⁇ ,” refers to non-coding sequences located within the retroviral genome which are required for encapsidation of retroviral RNA strands during viral particle formation, see e.g., Clever et al, 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.
• Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs.
• Self-inactivating (SIN) vectors refers to replication-defective vectors, e.g., in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
• the 3' LTR is modified such that the U5 region is replaced, for example, with an ideal poly(A) sequence.
• An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
• heterologous promoters examples include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
• SV40 viral simian virus 40
• CMV cytomegalovirus
• MoMLV Moloney murine leukemia virus
• RSV Rous sarcoma virus
• HSV herpes simplex virus
• modifications to the LTRs such as modifications to the 3' LTR, the 5' LTR, or both 3' and 5' LTRs, are also included.
• FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2.
• a retrovirus e.g., HIV-1 or HIV-2.
• Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et al, 2000, Cell, 101: 173.
• central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap.
• the DNA flap may act as a cis-active determinant of lentiviral genome nuclear import and/or may increase the titer of the virus.
• the retroviral or lentiviral vector backbones comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors.
• a transfer plasmid includes a FLAP element.
• a vector comprises a FLAP element isolated from HIV-1.
• a lentiviral vector contains a FLAP element with one or more mutations in the cPPT and/or CTS elements.
• a lentiviral vector comprises either a cPPT or CTS element.
• a lentiviral vector does not comprise a cPPT or CTS element.
• RNA export element refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
• RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al, 1991. J. Virol. 65: 1053; and Cullen et al, 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE).
• HCV human immunodeficiency virus
• RRE hepatitis B virus post-transcriptional regulatory element
• heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient
• vectors comprise a posttranscriptional regulatory element such as a WPRE or HPRE.
• vectors lack or do not comprise a posttranscriptional regulatory element such as a WPRE or HPRE.
• Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression.
• Illustrative examples of polyA signals that can be used in a vector includes an ideal polyA sequence (e.g., AATAAA, ATT AAA, AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a rabbit ⁇ -globin polyA sequence (r ⁇ gpA), or another suitable heterologous or endogenous polyA sequence known in the art.
• most or all of the viral vector backbone sequences are derived from a lenti virus, e.g., HIV-1.
• a lenti virus e.g., HIV-1.
• many different sources of retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein.
• a variety of lentiviral vectors are known in the art, see Naldini et al , (1996a, 1996b, and 1998); Zufferey et al, (1997); Dull et al , 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid.
• a retroviral vector comprises a left (5 ' ) lentiviral LTR; a Psi ( ⁇ ) packaging signal; a retroviral export element; a cPPT/FLAP; a promoter operably linked to a polynucleotide encoding a tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral LTR.
• the retroviral vector is preferably a lentiviral vector, more preferably an HIV lentiviral vector, and even preferably, an HIV-1 lentiviral vector.
• a lentiviral vector comprises a left (5 ' ) lentiviral LTR wherein the promoter region of the LTR is replaced with a heterologous promoter; a Psi ( ⁇ ) packaging signal; a retroviral export element; a cPPT/FLAP; a promoter operably linked to a polynucleotide encoding a tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral LTR.
• the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.
• a lentiviral vector comprises a left (5 ' ) lentiviral LTR; a Psi ( ⁇ ) packaging signal; a retroviral export element; a cPPT/FLAP; a promoter operably linked to a polynucleotide encoding a tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral LTR that comprises one or more modification compared to an unmodified LTR.
• the 3 ' LTR preferably comprises one or more deletions that prevent viral transcription beyond the first round of viral replication, more preferably comprises a deletion of the TATA box and Spl and NF- ⁇ transcription factor binding sites in the U3 region of the 3' LTR, and even more preferably is a self-inactivating (SIN) LTR.
• SIN self-inactivating
• a lentiviral vector comprises a left (5 ' ) lentiviral LTR wherein the promoter region of the LTR is replaced with a heterologous promoter; a Psi ( ⁇ ) packaging signal; a retroviral export element; a cPPT/FLAP; a promoter operably linked to a polynucleotide encoding a tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral SIN LTR.
• a lentiviral vector comprises a left (5 ' ) lentiviral LTR wherein the promoter region of the LTR is replaced with a heterologous promoter; a Psi ( ⁇ ) packaging signal; a retroviral export element; a cPPT/FLAP; a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter or transcriptionally active fragment thereof operably linked to a polynucleotide encoding a human tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral SIN LTR.
• a lentiviral vector comprises a left (5 ' ) lentiviral LTR wherein the promoter region of the LTR is replaced with a heterologous promoter; a Psi ( ⁇ ) packaging signal; a retroviral export element; a cPPT/FLAP; an elongation factor 1 alpha (EF1 ⁇ ) promoter or transcriptionally active fragment thereof operably linked to a polynucleotide encoding a human tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral SIN LTR.
• the EF1 ⁇ promoter lacks the first intron of the human EF l a gene and is referred to as an "EF1 ⁇ short promoter.” In other embodiments, the EF1 ⁇ promoter comprises the first intron of the human EF1 ⁇ gene and is referred to as an "EF1 ⁇ long promoter.”
• a lentiviral vector comprises a left (5 ' ) CMV promoter/HIV-1 chimeric LTR; a Psi ( ⁇ ) packaging signal; an RRE retroviral export element; a cPPT/FLAP; an MND promoter or EF l ⁇ -short promoter operably linked to a polynucleotide encoding a human tripeptidyl peptidase 1(TPP1) polypeptide; and a right (3 ' ) lentiviral SIN LTR.
• a lentiviral vector comprises a left (5 ' ) CMV promoter/HIV-1 chimeric LTR; a Psi ( ⁇ ) packaging signal; an RRE retroviral export element; a cPPT/FLAP; an MND promoter or EF l ⁇ -short promoter operably linked to a polynucleotide encoding a human tripeptidyl peptidase 1(TPP1) polypeptide; a right (3 ' ) lentiviral SIN LTR; and a heterologous polyadenylation signal.
• the polyadenylation signal is an artificial polyadenylation signal, a bovine growth hormone polyadenylation signal or a rabbit ⁇ -globin polyadenylation signal.
• Large scale viral particle production is often necessary to achieve a reasonable viral titer.
• Viral particles are produced by transfecting a transfer vector into a packaging cell that comprises viral structural and/or accessory genes, e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.
• the term "packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four or more viral structural and/or accessory genes.
• the packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known by those of skill in the art.
• a retroviral/lentiviral transfer vector can be introduced into a packaging cell line, via transfection, transduction or infection, to generate a producer cell or cell line.
• the packaging vectors can be introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection or electroporation.
• the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromy cin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.
• a selectable marker gene can be linked physically to genes encoding by the packaging vector, e.g., by IRES or self-cleaving viral peptides.
• Viral envelope proteins determine the range of host cells which can ultimately be infected and transformed by recombinant retroviruses generated from the cell lines.
• the env proteins include gp41 and gpl20.
• the viral env proteins expressed by packaging cells are encoded on a separate vector from the viral gag and pol genes, as has been previously described.
• retroviral -derived env genes which can be employed in particular embodiments include, but are not limited to: MLV envelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (Fowl plague virus), and influenza virus envelopes.
• RNA viruses e.g., RNA virus families of Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae) as well as from the DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) may be utilized.
• RNA viruses e.g., RNA virus families of Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae
• viruses include, but are not limited to, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.
• envelope proteins for pseudotyping a virus include, but are not limited to any of the following virus: Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), Influenza B, Influenza C virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus of the Norwalk virus group, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox,
• Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu)
• Influenza B Influenza C virus
• Hepatitis A virus Hepatitis B virus
• Hepatitis C virus Hepatitis D virus
• Hepatitis E virus Hepatitis E virus
• Rotavirus any virus of the Norwalk virus group
• enteric adenoviruses parvovirus
• Dengue fever virus Monkey pox
• Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus,
• Duvenhage virus European bat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as Argentine hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome
• packaging cells which produce recombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-G glycoprotein.
• lentiviral envelope proteins are pseudotyped with VSV-G.
• packaging cells are provided which produce recombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-G envelope glycoprotein.
• packaging cell lines is used in reference to cell lines that do not contain a packaging signal, but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which are necessary for the correct packaging of viral particles.
• Any suitable cell line can be employed to prepare packaging cells.
• the cells are mammalian cells.
• the cells used to produce the packaging cell line are human cells.
• Suitable cell lines which can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.
• the packaging cells are 293 cells, 293T cells, or A549 cells.
• the cells are A549 cells.
• the term "producer cell line” refers to a cell line which is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal.
• the production of infectious viral particles and viral stock solutions may be carried out using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, e.g., Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau ef al. (1992) J Virol. 66:5110- 5113. Infectious virus particles may be collected from the packaging cells using conventional techniques.
• the infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as is known in the art.
• the collected virus particles may be purified if desired. Suitable purification techniques are well known to those skilled in the art.
• host cells transduced with viral vector that expresses one or more polypeptides to generate genetically modified cells that are administered to a subject to treat and/or prevent and/or ameliorate at least one symptom of a neuronal ceroid lipofuscinoses can be found in, e.g., Kay, M. A. (1997) Chest 111(6 Supp.): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther.
• Lipidol. 11 179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7: 1744-52; Yang, N. S.
• a "host cell” includes cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide contemplated herein.
• Host cells may include packaging cells, producer cells, and cells infected with viral vectors.
• host cells infected with viral vector of the invention are administered to a subject in need of therapy.
• the term "target cell” is used interchangeably with host cell and refers to transfected, infected, or transduced cells of a desired cell type.
• the target cell is a stem cell or progenitor cell.
• the target cell is a somatic cell, e.g.
• the target cell is a hematopoietic cell, e.g. , a hematopoietic stem or progenitor cell, or CD34 + cell. Further therapeutic target cells are discussed, herein.
• cells are genetically modified to express a TPP1 polypeptide, and the genetically modified cells are used to treat neuronal ceroid lipofuscinoses.
• the cells may be genetically modified ex vivo, in vitro, or ex vivo.
• the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell.
• the terms, “genetically modified cells,” “modified cells,” and, “genetically engineered cells,” are used interchangeably.
• gene therapy refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects, or modifies expression of a gene, or for the purpose of expressing a therapeutic polypeptide, e.g., TPP1.
• the cells can be autologous/autogeneic ("self) or non-autologous ("non-self,” e.g. , allogeneic, syngeneic or xenogeneic).
• Autologous refers to cells from the same subject.
• Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
• Syngeneic refers to cells of a different subject that are genetically identical to the cell in comparison.
• Xenogeneic refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are allogeneic.
• vectors encoding TPP1 are introduced into one or more animal cells, preferably a mammal, e.g. , a non-human primate or human, and more preferably a human.
• a population of cells is transduced with a vector contemplated herein.
• the term "population of cells” refers to a plurality of cells that may be made up of any number and/or combination of homogenous or heterogeneous cell types, as described elsewhere herein.
• a population of cells may be isolated or obtained from umbilical cord blood, placental blood, bone marrow, or peripheral blood.
• a population of cells may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the target cell type to be transduced.
• hematopoietic stem or progenitor cells may be isolated or purified from a population of heterogeneous cells using methods known in the art.
• the cell is a primary cell.
• primary cell as used herein is known in the art to refer to a cell that has been isolated from a tissue and has been established for growth in vitro or ex vivo. Corresponding cells have undergone very few, if any, population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous cell lines, thus representing a more representative model to the in vivo state. Methods to obtain samples from various tissues and methods to establish primary cell lines are well-known in the art (see, e.g., Jones and Wise, Methods Mol Biol. 1997).
• Primary cells for use in the method of the invention are derived from, e.g., blood, lymphoma and epithelial tumors. In one embodiment, the primary cell is a hematopoietic stem or progenitor cell.
• stem cell refers to a cell which is an undifferentiated cell capable of (1) long term self -renewal, or the ability to generate at least one identical copy of the original cell, (2) differentiation at the single cell level into multiple, and in some instance only one, specialized cell type and (3) of in vivo functional regeneration of tissues.
• Stem cells are subclassified according to their developmental potential as totipotent, pluripotent, multipotent and oligo/unipotent.
• Self-renewal refers a cell with a unique capacity to produce unaltered daughter cells and to generate specialized cell types (potency). Self- renewal can be achieved in two ways.
• Asymmetric cell division produces one daughter cell that is identical to the parental cell and one daughter cell that is different from the parental cell and is a progenitor or differentiated cell.
• Symmetric cell division produces two identical daughter cells.
• "Proliferation” or “expansion” of cells refers to symmetrically dividing cells.
• progenitor or “progenitor cells” refers to cells have the capacity to self-renew and to differentiate into more mature cells. Many progenitor cells differentiate along a single lineage, but may have quite extensive proliferative capacity.
• Hematopoietic stem cells give rise to committed hematopoietic progenitor cells (HPCs) that are capable of generating the entire repertoire of mature blood cells over the lifetime of an organism.
• HPC hematopoietic stem cell
• myeloid e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
• lymphoid lineages e.g., T- cells, B-cells, NK-cells
• the HSC is a CD34 + cell.
• hematopoietic stem and progenitor cells When transplanted into lethally irradiated animals or humans, hematopoietic stem and progenitor cells can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell pool.
• Preferred target cell types transduced with the compositions and methods contemplated herein include, hematopoietic cells, preferably human hematopoietic cells, more preferably human hematopoietic stem and progenitor cells, and even more preferably CD34 + human hematopoietic stem cells.
• Illustrative sources to obtain hematopoietic cells transduced with the methods and compositions contemplated herein include, but are not limited to: cord blood, bone marrow or mobilized peripheral blood.
• hematopoietic cells transduced with viral vectors encoding TPP1 contemplated herein include CD34 + cells.
• CD34 + cell refers to a cell expressing the CD34 protein on its cell surface.
• CD34 refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell- cell adhesion factor.
• CD34 + is a cell surface marker of both hematopoietic stem and progenitor cells.
• hematopoietic stem or progenitor cells suitable for transduction with the methods and compositions contemplated herein include hematopoietic cells that are CD34 + CD38 Lo CD90 + CD45 RA- hematopoietic cells that are CD34 + CD59 + , Thyl/CD90 + CD38 Lo/- , C-kit/CD117 + , and Lin (-) and hematopoietic cells that are CD133 + .
• hematopoietic cells transduced with viral vectors encoding
• TPP1 contemplated herein include CD34 + CD133 + cells.
• the SLAM (Signaling lymphocyte activation molecule) family is a group of >10 molecules whose genes are located mostly tandemly in a single locus on chromosome 1 (mouse), all belonging to a subset of immunoglobulin gene superfamily, and originally thought to be involved in T-cell stimulation.
• This family includes CD48, CD150, CD244, etc., CD150 being the founding member, and, thus, also called slamFl, i.e. , SLAM family member 1.
• the signature SLAM code for the hematopoietic hierarchy is hematopoietic stem cells (HSC) - CD150 + CD48 " CD244 " ;
• MPPs multipotent progenitor cells
• LRPs lineage-restricted progenitor cells
• CMP common myeloid progenitor
• GMP common myeloid progenitor
• GMP granulocyte-macrophage progenitor
• MMP megakaryocyte-erythroid progenitor
• MEP megakaryocyte-erythroid progenitor
• hematopoietic cells transduced with viral vectors encoding
• TPP1 contemplated herein include CD150 + CD48 " CD244- cells.
• a population of hematopoietic cells comprising hematopoietic stem and progenitor cells (HSPCs) transduced with a viral vector encoding TPP1 as contemplated herein is provided.
• the HSPCs are CD34 + hematopoietic cells.
• compositions and formulations contemplated herein may comprise a combination of any number of transduced or non-transduced cells or a combination thereof, viral vectors, polypeptides, and polynucleotides contemplated herein.
• compositions include, but are not limited to pharmaceutical compositions.
• composition refers to a composition formulated with a
• compositions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
• the compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
• agents such as, e.g., cytokines, growth factors, hormones, small molecules, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
• additional agents do not adversely affect the ability of the composition to deliver the intended therapy.
• compositions contemplated herein may comprise a combination of transduced or non-transduced cells or a combination thereof, and viral vectors formulated with a pharmaceutically-acceptable carrier for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
• compositions contemplated herein may comprise a combination of viral vectors formulated with a pharmaceutically-acceptable carrier for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
• compositions contemplated herein comprise a population of cells, comprising a therapeutically-effective amount of transduced cells, e.g., hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, CD133 + cells, etc., formulated with one or more pharmaceutically acceptable carriers.
• transduced cells e.g., hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, CD133 + cells, etc.
• compositions comprising a retroviral vector, e.g., a lentiviral vector formulated with one or more pharmaceutically acceptable carriers.
• Pharmaceutical compositions contemplated herein comprise transduced cells comprising a vector or provirus encoding TPP1 as contemplated herein and a pharmaceutically acceptable carrier.
• phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic cells are administered.
• pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject.
• a composition comprising a carrier is suitable for parenteral administration, e.g. , intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
• compositions contemplated herein comprise genetically modified hematopoietic stem and/or progenitor cells and a
• compositions comprising a cell-based composition contemplated herein can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals
• the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition.
• the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc. , when combined with other components of the composition.
• the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g. , pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g.
• a lubricant e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.
• a disintegrant e.g. , starch, sodium starch glycolate, etc.
• a wetting agent e.g. , sodium lauryl sulfate, etc.
• compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins,
• hydroxymethylcelluloses polyvinylpyrrolidones and the like.
• Such carrier solutions also can contain buffers, diluents and other suitable additives.
• buffer refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
• buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
• PBS Dulbecco's phosphate buffered saline
• D5W 5% dextrose in water
• normal/physiologic saline (0.9% NaCl).
• the pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7.
• the composition has a pH in a range from about 6.8 to about 7.4, e.g. , 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.
• the composition has a pH of about 7.4.
• compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium.
• the compositions may be a suspension.
• the term "suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
• compositions contemplated herein are formulated in a suspension, where the hematopoietic stem and/or progenitor cells are dispersed within an acceptable liquid medium or solution, e.g. , saline or serum-free medium, in an intravenous (IV) bag or the like.
• acceptable liquid medium or solution e.g. , saline or serum-free medium
• IV intravenous
• Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium.
• a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of cells, e.g., hematopoietic stem and progenitor cells.
• the therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
• compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects.
• the pharmaceutically acceptable cell culture medium is a serum free medium.
• Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of
• the serum-free medium is animal-free, and may optionally be protein-free.
• the medium may contain biopharmaceutically acceptable recombinant proteins.
• Animal-free medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal-free medium and the nutrients are obtained from synthetic, plant or microbial sources.
• Protein-free medium in contrast, is defined as substantially free of protein.
• serum-free media used in particular compositions includes, but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.
• compositions comprising hematopoietic stem and/or progenitor cells are formulated in PlasmaLyte.
• compositions comprising hematopoietic stem and/or progenitor cells are formulated in a cryopreservation medium.
• cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
• cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS 10, CryoStor CS5, and CryoStor CS2.
• compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS 10.
• composition is substantially free of
• endotoxin mycoplasma, endotoxin, and microbial contamination.
• substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
• compositions comprising hematopoietic stem or progenitor cells transduced with a retroviral vector contemplated herein contains about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL, 3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.
• compositions and formulations suitable for the delivery of viral vector systems are contemplated including, but not limited to, retroviral (e.g. , lentiviral) vectors.
• Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate,
• Liposomes as described in greater detail below, are lipid bilayers entrapping a fraction of aqueous fluid. DNA spontaneously associates to the external surface of cationic liposomes (by virtue of its charge) and these liposomes will interact with the cell membrane.
• formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g. , enteral and parenteral, e.g. , intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and
• the genetically modified cells contemplated herein provide improved drug products for use in the prevention, treatment, and amelioration of a neuronal ceroid fuscinoses or for preventing, treating, or ameliorating at least one symptom associated with a neuronal ceroid fuscinoses or a subject having a mutation in a TPPl gene that decreases or abolishes TPPl expression.
• the neuronal ceroid fuscinoses is late-infantile neuronal ceroid fuscinoses (LINCL).
• the neuronal ceroid fuscinoses is juvenile Batten disease (JNCL).
• the term "drug product” refers to genetically modified cells produced using the compositions and methods contemplated herein.
• the drug product comprises genetically modified hematopoietic stem or progenitor cells, e.g., CD34 + cells.
• CD34 + cells genetically modified hematopoietic stem or progenitor cells
• a viral vector of the invention comprises an expression control sequence that expresses a therapeutic transgene encoding a polypeptide that provides curative, preventative, or ameliorative benefits to a subject diagnosed with or that is suspected of having an NCL, LINCL, JNCL, or a subject having TPP1 gene comprising one or more mutations that decrease TPP1 expression.
• the retroviral vectors are administered by direct injection to a cell, tissue, or organ of a subject in need of gene therapy, in vivo.
• cells are transduced in vitro or ex vivo with vectors contemplated herein, and optionally expanded ex vivo. The transduced cells are then administered to a subj ect in need of gene therapy .
• Cells suitable for transduction and administration in the gene therapy methods contemplated herein include, but are not limited to stem cells, progenitor cells, and differentiated cells as described elsewhere herein.
• the transduced cells are hematopoietic stem or progenitor cells as described elsewhere herein.
• Preferred cells for use in the gene therapy compositions and methods contemplated herein include autologous/autogeneic ("self) cells.
• a subject includes any animal that exhibits symptoms of a neuronal ceroid lipofuscinoses that can be treated with the gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein.
• Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
• Non-human primates and, preferably, human patients, are included.
• Typical subjects include human patients that have an NCL, have been diagnosed with an NCL, or are at risk or having an NCL.
• the term "patient” refers to a subject that has been diagnosed with a particular disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.
• treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
• prevention and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
• the phrase "ameliorating at least one symptom of refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated.
• the disease or condition being treated is an NCL, wherein the at least one symptom is selected from the group consisting of: progressive loss of motor function, progressive loss of cognitive function, visual impairment, blindness, speech difficulties, ataxia, dementia, heart problems, behavioral problems, difficulty sleeping, and problems with attention, and seizures.
• a subject is administered an amount of genetically modified cell or gene therapy vector sufficient to treat, prevent, or ameliorate at least one symptom of an NCL.
• the term “amount” refers to "an amount effective” or “an effective amount” of a virus or transduced therapeutic cell to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
• a “prophylactically effective amount” refers to an amount of a virus or transduced therapeutic cell effective to achieve the desired prophylactic result.
• the prophylactically effective amount is less than the therapeutically effective amount.
• a “therapeutically effective amount” of a virus or transduced therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects.
• the term “therapeutically effective amount” includes an amount that is effective to "treat" a subject (e.g. , a patient).
• an important advantage provided by the vectors, compositions, and methods of the present invention is the high efficacy of gene therapy that can be achieved by administering populations of cells comprising high percentages of transduced cells compared to existing methods.
• transduced cells may be administered as part of a bone marrow or cord blood transplant in an individual that has or has not undergone bone marrow ablative therapy.
• transduced cells of the invention are administered in a bone marrow transplant to an individual that has undergone chemoablative or radioablative bone marrow therapy.
• a dose of transduced cells is delivered to a subject intravenously.
• transduced hematopoietic stem cells are intravenously administered to a subject.
• the effective amount of transduced cells provided to a subject is at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, or at least 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
• the effective amount of transduced cells provided to a subject is about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x
• 10 6 cells/kg about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, or about 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
• the effective amount of transduced cells provided to a subject is from about 2 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 4 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 5 x 10 6 cells/kg to about 10 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/
• a pharmaceutical composition comprising the genetically modified cells described herein may be administered at a dosage of 10 2 to 10 10 cells/kg body weight, preferably 10 5 to 10 7 cells/kg body weight, including but not limited to 1 x 10 6 cells/mL, 2 x 10 6 cells/mL, 3 x 10 6 cells/mL, 4 x 10 6 cells/mL, 5 x 10 6 cells/mL, 6 x 10 6 cells/mL, 7 x 10 6 cells/mL, 8 x 10 6 cells/mL, 9 x 10 6 cells/mL, 10 x 10 6 cells/mL, and all integer values within those ranges.
• the number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.
• the cells are generally in a volume of a liter or less, can be 500 mLs or less, even 250 mLs or 100 mLs or less.
• the density of the desired cells in particular embodiments is typically greater than 10 6 cells/mL, 10 7 cells/mL, or 10 8 cells/mL.
• the clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells.
• Cell-based compositions may be administered multiple times at dosages within these ranges.
• the cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.
• the drug product is administered once.
• the drug product is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 year, 2 years, 5, years, 10 years, or more.
• myeloproliferative sarcoma virus enhancer negative control region deleted, dl587rev primer-binding site substituted (MND) promoter or a short elongation factor 1 alpha (EF1 ⁇ ) promoter; a polynucleotide encoding a tripeptidyl peptidase 1(TPP1) polypeptide; and a self-inactivating (SIN) 3 ' LTR were constructed. See e.g., Figure 1 and SEQ ID NOs: 1 and 2. Tables 1 and 2 show the Identity, Genbank Reference, Source Name and Citation for the various nucleotide segments of exemplary lentiviral vectors encoding TPPl .
• TPPl 27X/R208X; TPP 1 v" cells Human fibroblasts deficient in TPPl activity because of homozygous mutations in the TPP 1 gene (Rl 27X/R208X; TPP 1 v" cells) were acquired from the Coriell Institute Cell Repository (cell line GM16485).
• TPPl "7" cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) for twenty -four hours prior to transduction. Cultured TPPl "7" cells were resuspended at 5.0E4 cells/mL of DMEM plus 10% FBS and two mL of this cell suspension were plated per well in a 6-well tissue culture plate and placed at 37°C.
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• Frozen cell pellets from wild type control cells, ⁇ 1 -/- cells, and ⁇ 1 -/- cells transduced with the lentiviral vectors encoding TPP1 were thawed on ice for Western blotting.
• Pellets were resuspended by pipetting gently up and down and cells were incubated for 10 minutes at room temperature on a plate rocker.
• Loading dye was prepared by adding 25 ⁇ L. ⁇ -mercaptoethanol to 475 ⁇ L. 4X Laemmli sample buffer (Bio-Rad). Samples were mixed in a 3: 1 sample to loading dye ratio with 30 ⁇ L. prepared loading dye to 90 ⁇ L. sample. 20 ⁇ L. of each sample and 8 ⁇ L Precision Plus Protein Kaleidoscope ladder were loaded into the wells of a NuPage 4-12 Bis-Tris protein gel. Gels were run in IX MES SDS running buffer for 40 minutes at 200V.
• Membranes were incubated for one hour at room temperature in secondary antibody cocktail and rinsed three times with Tris-buffered saline for five minutes at room temperature. Blots were imaged on a Licor Odyssey CLX imaging system.
• TPP- 1 activity Fluorometric measurement of TPP- 1 activity was calculated based on cleavage of the Ala-Ala-Phe-7-Amido-4-methylcoumarin substrate (AAF-MCA) as described previously, with some modifications (Page et al. , 1993. Arch Biochem
• Figure 3 A shows the results from a representative experiment assaying TPPl enzymatic activity in wild type control cells, ⁇ 1 -/- cells, and ⁇ 1 -/- cells transduced with the lentiviral vectors encoding TPPl (pMND-TPPl and pEFl ⁇ -TPPl).
• TPPl overexpression in transduced cells also led to secretion of enzymatically active TPPl in the cell culture supernatant.
• TPPl activity in both patient and wild type fibroblasts remained at background levels; whereas overexpression of TPPl in transduced TPP ⁇ " fibroblasts increased TPPl activity in the supernatant 10-fold.
• Figure 3B Thus, TPPl gene therapy not only corrects transduced cells, but also has the potential to correct TPPl deficiency in neighboring cells.
• TPPl deficient patient neurons progressively accumulate storage material with classical NCL features, and have additional morphological changes in lysosomal and endoplasmic reticulum compartments (Lojewski et al, HMG, 2014, v23, pp2005-2022).
• TPPl enzyme activity levels will be determined, and neuronal morphology will be assessed, e.g., to determine the size and number of storage deposits (using established autofluorescence and subunit c immunostaining procedures).
• TPPl -/- induced pluripotent stem cells were differentiated into neurons.
• the neurons from patients with confirmed CLN2 mutations were transduced at an MOI of 5 with lentiviral vectors comprising an MND or EF l ⁇ -short promoter operably linked to a polynucleotide encoding TPPl.
• Transduced cells were fixed and imaged using confocal microscopy at 17 days post-transducti on/post-differentiation, as neural progenitors and as differentiated neurons.
• Antibodies specific to the LLV expressed protein, TPPl, and the CLN2 lipofuscin storage material, ATP synthase subunit c were used as co-stains to visualize the two simultaneously in the same cells.
• mice with TPPl mutations will be administered HSCs transduced with lentiviral vectors encoding TPPl and phenotypically characterized.
• TPPl mutant mice will undergo treatment to ablate bone marrow hematopoietic stem cells and administered HSCs transduced with lentiviral vectors encoding TPPl at no more than 2 weeks of age.
• mice will undergo clinical assessment, which includes observation for tremors, general body condition, weight gain (weekly, starting at ⁇ 4 weeks of age), grip strength (biweekly, beginning at ⁇ 8 weeks of age), rotarod (at -13, 18 weeks of age), and gait analysis (at -16 and -24 weeks of age).
• mice will be tested post-transplant for other parameters to assess their general health and immune system reconstitution after hematopoietic stem cell therapy including full clinical blood chemistry panels, CNS gross morphology and histological analysis to assess storage material, neuronal and glial cell numbers, and morphology (e.g., axonal degeneration) in sagittal sections (to capture multiple brain regions in each section), evidence of cross-correction (expression) in tissues affected by TPPl deficiency, TPPl enzyme activity in blood/brain/tissue ly sates, bone marrow morphology, measurement of vector copy number in mouse bone marrow at the end of all experiments; and identification of engrafted cells.
• CNS gross morphology and histological analysis to assess storage material
• neuronal and glial cell numbers e.g., axonal degeneration
• morphology e.g., axonal degeneration
• sagittal sections to capture multiple brain regions in each section
• Human CD34+ cells were transduced with a lentiviral vector (LVV) comprising an EF1 ⁇ promoter or MND promoter operably linked to a polynucleotide encoding CLN2.
• LVV lentiviral vector
• Cells were prestimulated in cytokine containing media for 48 hours and transduced for 24 hours at an MOI of either 5 or 15 using either standard transduction conditions of 8 ⁇ g/mL protamine sulfate (PS) or 200 ⁇ g/mL F108 (poloxamer 338, BASF) and 10 ⁇ PGE2 (Cayman). After transduction, cells were plated in methylcellulose and cultured for approximately 14 days to allow for hematopoietic progenitor colony formation. Colonies were pooled for VCN analysis.
• VCNs Transduction in protamine sulfate yielded VCNs below 1.5 and VCN increased with increasing MOI. Substantial increases in VCN were observed in cells transduced in the presence of F 108 and PGE2 at either MOI ( Figure 4, top left panel). After transduced cells were cultured in cytokines for 7 days; cell pellets and supernatants were assayed for TPP-1 activity. TPP-1 activity was higher in cells with higher VCNs, i.e., cells transduced in the presence of F 108 and PGE2 ( Figure 4, top middle and top right panels).
• VCN was increased in cytokine cultured cells at day 7 at all MOIs of 5, 15, and 30 ( Figure 4, bottom left panel). Individual colonies were plucked and analyzed by qPCR for VCN from mouse hematopoietic progenitors; F108 and PGE2 increased VCN in these cells ( Figure 4, lower middle panel). F 108 and PGE2 also substantially increased transduction efficiency ( Figure 4, lower right panel).
• Cells were prestimulated in cytokine containing media for 48 hours and transduced for 24 hours at an MOI of either 5, 15, or 30 using either standard transduction conditions of 8 ⁇ g/mL protamine sulfate (PS) or 200 ⁇ g/mL F108 (poloxamer 338, BASF) and 10 ⁇ PGE2 (Cayman). After transduction, cells were plated in methylcellulose and cultured for approximately 14 days to allow for hematopoietic progenitor colony formation or cultured in cytokine containing media for 14 days. Cell growth, VCN from liquid cultures, individual colony VCN and %LVV+ cells from Day 14 methylcellulose cultures, and TPP1 activity were measured. Cell growth was not adversely affected by transducing the CD34 + cells with lentiviral vector in the presence of F 108 and PGE2. Figure 5.
• VCN was measured in transduced cells cultured in cytokines at 7 days and 14 days.
• F 108 and PGE2 increased transduction across all MOIs for both lentiviral vectors.
• Figure 6
• TPP-1 activity was higher in cells with higher VCNs.

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  • 2017-06-13 WO PCT/US2017/037230 patent/WO2017218519A1/en not_active Ceased
  • 2017-06-13 EP EP17813925.9A patent/EP3468594A4/en not_active Withdrawn
  • 2017-06-13 CA CA3058735A patent/CA3058735A1/en not_active Abandoned
  • 2017-06-13 US US16/309,412 patent/US20220323612A1/en not_active Abandoned
  • 2017-06-13 CN CN201780043899.7A patent/CN109475619A/zh active Pending
  • 2017-06-13 AU AU2017283474A patent/AU2017283474A1/en not_active Abandoned
  • 2017-06-13 JP JP2019517200A patent/JP2019517281A/ja active Pending

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