Title:  Combinations and methods for promoting in vivo liver cell proliferation and enhancing in vivo liver-directed gene transduction

United States Patent:  6,248,725

Assignee:  Amgen, Inc. (Thousand Oaks, CA)

Filed:  February 23, 1999

Combinations and methods for inducing a semi-synchronous wave of liver cell proliferation in vivo and combinations and methods for inducing a semi-synchronous wave of liver cell proliferation and achieving transduction of proliferating liver cells in vivo are disclosed.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "organ" means a discrete group of cells related by function, as may be used, for example, for transplantation or in a life support system, or the like.

As used herein, time is measured from the first administration of a growth factor to the individual, and is considered "hour 0" of "day 1."

As used herein, "semi-synchronous" means that although the cells that are induced to proliferate do not enter S-phase at exactly the same time, they do so relatively contemporaneously, such that the first induced cell divisions and the last induced cell divisions occur within a period of between about 0 hours and about 28 days, depending on the species, weight, and other variables affecting induced liver cell proliferation and the quantity and the regimen of factor administration.

As used herein, "tri-iodothyronine" or "T3" refers to thyroid hormone or its analogs, and also includes compounds that bind and activate the thyroid hormone nuclear receptor.

The timing, dosage and mode of T3 administration should be determined by the prescribing physician or veterinarian, and may vary depending on mode of administration, the species, age, and condition of the individual and in accordance with the needs of the individual and the time schedule of administration of the other factors. T3 may administered at any effective time before entrance of liver cells into S-phase is desired. T3 administration may also continue thereafter. Generally, however, T3 may be administered between about 0 and about 28 days before entrance of liver cells into S-phase is desired. Preferably T3 is administered between about 6 hours and about 14 days before entrance of liver cells into S-phase is desired. Most preferably, T3 is administered between about 24 hours and about 8 days before entrance of liver cells into S-phase is desired. More than one administration of T3 may be desirable in accordance with the needs of the individual as determined by the prescribing physician or veterinarian, and T3 may be administered any effective amount of times. Successive administrations generally can be performed at intervals ranging from about hourly to about weekly, but are preferably done at about daily intervals, or by continuous infusion. T3 administration may continue for any effective time after liver cells have begun to proliferate.

T3 may be administered in any effective daily dosage. In general, however, daily dosages will range from about 80 .mu.g of T3 per kg of body weight per day (.mu.g/kg) to about 80 mg/kg. Preferably, T3 is administered in daily dosages ranging from about 400 .mu.g/kg to about 40 mg/kg. Most preferred is the administration of daily dosages of about 4 mg/kg of T3. T3 may be administered by any effective route. It may be administered, for example, intravenously ("IV"), intramuscularly ("IM"), intraperitoneally, directly into the liver, subcutaneously, orally, inhaled, or by suppository. Subcutaneous administration is preferred. For subcutaneous administration, T3 may be dissolved in an appropriate volume of 0.01 M NaOH in 0.9 M NaCl, or any other effective carrier, and administered as a bolus. The formulation of T3 for administration is well known in the art.

As used herein, "keratinocyte growth factor" or "KGF" refers to the keratinocyte growth factor polypeptide or one of its analogs, or alternatively an active fragment of keratinocyte growth factor or one of its analogs, or a factor that binds and activates the keratinocyte growth factor receptor. Preferred is the recombinantly produced form of KGF. See, e.g., U.S. Pat. No. 5,731,170, and PCT Application No. WO 90/08771, published Aug. 9, 1990 (directed to full length forms of KGF and variants); and PCT Application No. WO 96/11949, published Apr. 25, 1996; PCT Application No. WO 96/11951, published Apr. 25, 1996; and PCT Application No. WO 98/24813, published Jun. 11, 1998 (directed to stable analogs of KGF) all of which are incorporated herein by reference in their entirety, including figures.

The timing, dosage and mode of KGF administration should be determined by the prescribing physician or veterinarian, and may vary depending on the mode of administration, the species, age, and condition of the individual, and in accordance with the needs of the individual and the time schedule of administration of the other factors. KGF may administered at any effective time before entrance of liver cells into S-phase is desired. KGF administration may also continue thereafter. Generally, however, KGF may be administered between about 0 and about 28 days before entrance of liver cells into S-phase is desired. Preferably KGF is administered between about 6 hours and about 14 days before entrance of liver cells into S-phase is desired. Most preferably, KGF is administered as a single bolus between about 24 hours and about 8 days before entrance of liver cells into S-phase is desired. More than one administration of KGF may be desirable in accordance with the needs of the individual as determined by the prescribing physician or veterinarian, and KGF may be administered any effective amount of times. Successive administrations generally can be performed at intervals ranging from about hourly to about weekly, but are preferably done at about daily intervals, or by continuous infusion. KGF administration may continue for any effective time after liver cells have begun to proliferate, but preferably for not longer than about 12 days thereafter. KGF may be administered in any effective daily dosage. In general, however, daily dosages ranging from about 5 .mu.g/kg to about 20 mg/kg. Preferably, KGF is administered in daily dosages ranging from about 100 .mu.g/kg to about 10 mg/kg. Most preferred is the administration of daily dosages of about 1 mg/kg of KGF. KGF may be administered by any effective route. It may be administered, for example, IV, IM, intraperitoneally, directly into the liver, subcutaneously, orally, by suppository, or by production in situ in the liver after liver transduction with an effective vector with a gene construct for KGF expression, or the like, for example an adenovirus vector, or the like. Subcutaneous administration as a bolus is preferred. For subcutaneous administration, KGF may be dissolved in any effective buffer. For example lyophilized KGF may be reconstituted in a 0.01% by weight polyoxyethylenesorbitan monolaurate solution (Sigma) (TWEEN 20), or any other effective buffer. Other suitable buffers are well known in the art.

A triple combination of T3, KGF and hepatocyte growth factor (HGF) was also tried. The cell proliferating characteristics, and the transduction efficiency of the triple combination was not statistically significantly different from the T3/KGF combination, although in some experiments the total number of cells induced to proliferate and liver cells transduced may have been slightly higher.

In accordance with the present invention, the factors may be administered in any effective order or time interval. However, the factors are preferably "concurrently administered," meaning that independent of the order in which the factors are administered, the factors are administered within a time interval such that the effect of the factors on the proliferation of liver cells is at least greater than additive. The factors may also be administered together. Preferably, dividing liver cells in a quantity about 10% or greater than the sum of the quantity of cells that would have resulted had the factors been independently administered will result from concurrent administration. Most preferably, dividing liver cells in a quantity about 30% or greater than the sum of the quantity of liver cells that would have resulted had the factors been independently administered will result from the concurrent administration.

In accordance with the present invention, each factor may be independently administered any effective number of times, including more than once, as may be indicated by a physician or veterinarian.

In accordance with the present invention, the factors may be administered in any effective amount. However, preferably administration of an "effective amount" will be an amount of each factor such that when administered in combination with the other factor or factors the effect of the factors on the induction of in vivo liver cell proliferation is synergistic. In accordance with the present invention, the amount of each factor administered is such that the effect of the factors on the proliferation of liver cells is at least greater than additive. Preferably, proliferating liver cells in a quantity about 10% or greater than the sum of the quantity of liver cells that would have resulted had the factors been independently administered in those amounts will result from the concurrent administration of an effective amount of each factor. Most preferably, proliferating liver cells in a quantity about 30% or greater than the sum of the quantity of liver cells that would have resulted had the factors been independently administered in those amounts will result from the concurrent administration of an effective amount of each factor.

The utility of the administration of any particular amount of each factor and the time and regimen for the administration of the different factors will vary according to several variables, including the species, weight, and other variables, and must be evaluated empirically. It is within the skill of the art to modify the procedure and produce the desired effect in a specific subject.

Several liver-directed gene transfer vectors may be used in accordance with the present invention, including retroviral vectors, adeno-associated virus vectors, lipofectant agents, receptor based transfer, and combinations thereof. See Ferry & Heard, Human Gene Ther. 9:1975-1981 (1998). Preferred are retroviral vectors. The production and use of retroviral vectors as gene transfer vectors is well documented in the literature, including the articles by Mann, et al., Cell 33:153-159 (1983), Cosset, et al., J. Virol. 69(12):7430-7436 (1995), which are hereby incorporated by reference in their entirety. Production of retroviral vector as described by Cosset, et al., is preferred. Most preferred is the administration of a retrovirus complexed with cationic liposomes, in particular the cationic liposome DiOctadecylamidoGlycylSpermine (DOGS). Infectivity of retroviral vectors has been found to be increased by the addition of DOGS to the retroviral vector preparations.

In accordance with the present invention, a liver-directed vector is preferably administered such that it transduces the liver cells within a few hours of maximal liver-cell division. However, the vector may be administered prior to or after that time, as may be more effective, depending on the mode of administration of the vector, the species, age, condition, and in accordance with the needs of the individual as determined by the prescribing physician or veterinarian. Generally, however, the vector may be administered between about 0 and about 28 days after administration of the factors. Preferably the vector is administered between about 6 hours and about 14 days after factor administration. Most preferably, the vector is administered between about 24 hours and about 8 days after factor administration. More than one administration of vector may also be desirable in accordance with the needs of the individual as determined by the prescribing physician or veterinarian. These successive administrations generally can be performed at intervals ranging from hourly to weekly, but are preferably done at between about 6 and about 24 hour intervals.

The method of administering the liver-directed vector to the liver contemplated in accordance with the present invention will vary in accordance with the needs of the individual as determined by the physician or veterinarian. In general, however, in vivo liver transduction may be performed according to a modified method described in Rettinger, et al., J. Surg. Res. 54:418-425 (1993), which is hereby incorporated by reference in its entirety. The modified method of Rettinger is preferred. Subjects are anesthetized, and a laparotomy is performed. The portal vein and hepatic artery are clamped using micro-aneurysm clips, or the like, and the portal vein is cannulated with a needle having an appropriate gauge (according to the size of the subject) proximal to the clip and retroviral supernatant is injected. Hemostasis may be achieved by direct pressure to the portal vein and topical thrombin application, or the like. The clips are removed and the abdomen sutured, and the subjects are allowed to convalesce. See Forbes, et al., Gene Therapy 5:552-555 at 554 (1998), which is hereby incorporated by reference in its entirety.

Alternatively, the procedure may be accomplished with portal injection of the liver-directed vector, in which case the portal vein is cannulated and the vector solution pumped into the portal vein over a period of time. See, e.g., Kay, et al., Human Gene Therapy 3:641-647 at 642 (1992) (using 1 ml of viral supernatant in 8 .mu.g/ml Polybrene infused over 50 minutes in mice), which is hereby incorporated by reference in its entirety.

Alternatively, the vector is delivered by asanguineous perfusion of the liver, whereby the liver is selectively perfused after excluding normal blood flow from the liver. This may be accomplished by clamping the hepatic artery, portal vein, suprahepatic vena cava, right suprarenal vein and infrahepatic vena cava to achieve the total vascular exclusion of the liver. The portal vein is then cannulated with a catheter of appropriate gauge, while an incision is made in the anterior wall of the infrahepatic vena cava, with a suction cannula placed into the vena cava to collect outflow. Liver perfusion, preferably single pass perfusion, is then performed using a pump. Flow rate may range from 0.1 ml/min per gram of liver to 10 ml/min per gram of liver. A rate of 1 ml/min per gram of liver is preferred, and may be increased to ensure satisfactory perfusion of the organ. After perfusion, the portal and caval vein incisions are sutured and the liver is revascularized. Cardoso, et al., Human Gene Therapy 4:411-418 at 412 (1993) , which is hereby incorporated by reference in its entirety.

Alternatively, another preferred method of vector administration is by peripheral vein intravenous injection. The vector may be administered by intravenous injection any number of effective times, including more than once, as may be indicated by a physician or veterinarian. The intravenous injection may be administered at any effective time, as described supra, but is most preferably administered 18-36 hours after the administration of the growth factors.

Infectivity of liver-directed vectors, in particular retroviral vectors, and hepatic transfection efficiency can be increased by the addition of liposomes, preferably cationic liposomes, to the liver-directed vector preparations. The cationic liposome DiOctadecylamidoGlycylSpermine (DOGS) is preferred. DOGS is effective in concentrations of about 1 to about 30 .mu.g per ml. Preferred is a final DOGS concentration of about 5 .mu.g per ml. The titer of vector/DOGS solution administered has preferably about 1x10⁹ i.u./ml after ultrafiltration.

Any effective volume of vector may be administered. The volume to be administered will depend on the i.u./ml of vector preparation, and the total number of i.u. it is desired to administer. In general, however, given a vector preparation having 1x10⁹ i.u./ml, about 0.1 ml of vector preparation per kg of subject weight to about 80 ml of vector preparation per kg of subject weight may be administered. Most preferred is the administration of about 8 ml of a solution of 1x10⁹ i.u./ml per kg of subject weight. Although any effective amount of total i.u. may be administered, preferably a total of about 8x10⁹ i.u. per kg of subject weight are administered.

Examples of conditions which are effectively treated with the combinations and methods of the present invention include treatment and prevention of any conditions that may be prevented or ameliorated by the proliferation of liver cells, including liver cell deficiencies, cirrhosis of the liver, and the like. Other conditions which are effectively treated with the combinations and methods of the present invention include treatment and prevention of any conditions that may be prevented or ameliorated by the expression in the liver of RNAs, polypeptides or proteins, or combinations thereof, for action in the liver or secretion of polypeptides or proteins into the bloodstream or the gastrointestinal tract.

The liver directed vector includes a nucleic acid engineered such that it encodes the RNA, protein or polypeptide to be expressed, operably linked to a sequence, such as a promoter or the like, and alternatively also an enhancer or the like, such that the encoded RNA, or mRNA for the polypeptide or protein, will be transcribed in the liver cells. If a protein or polypeptide is to be expressed, the nucleic acid is preferably engineered such that when the mRNA is transcribed it has a sequence such that it may be translated. See, e.g., I11, et al., Blood Coagulation and Fibrinolysis 8(Suppl 2):S23-S30 (1997)(incorporated herein by reference in its entirety).

The methods and compositions described herein find particular utility in the expression in the liver of RNAs, proteins and polypeptides known to have a therapeutic effect, including, but not limited to, for example, afamin (see, e.g., U.S. Pat. Nos. 5,652,352 and 5,767,243, hereby incorporated by reference including drawings); anti-inflammatory CD14 peptides (see, e.g., U.S. Pat. No. 5,766,593, hereby incorporated by reference including drawings); art (see, e.g., U.S. Pat. No. 5,766,877, hereby incorporated by reference including drawings); erythropoietins (see, e.g., U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, 5,621,080 and 5,756,349, hereby incorporated by reference including drawings); granulocyte colony-stimulating factor (see, e.g., United States Patent Nos. hereby incorporated by reference including drawings); granulocyte-colony stimulating factors (see, e.g., U.S. Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823 (human) and U.S. Pat. No. 5,606,024 (canine) and PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including drawings); interferon consensus (see, e.g., U.S. Pat. Nos. 5,661,009, 5,372,808, 5,541,293, 4,897,471, and 4,695,623, hereby incorporated by reference including drawings); interleukins (see, e.g., U.S. Pat. No. 5,075,222, hereby incorporated by reference including drawings); leptin (OB protein) (see. e.g., PCT Publication Nos. 96/40912, 96105309, 97/00128, 97/01010 and 97/06816, hereby incorporated by reference including drawings); NDF peptides (see, e.g., U.S. Pat. No. 5,670,342, hereby incorporated by reference including drawings); platelet-derived growth factor B (see, e.g., U.S. Pat. Nos. 5,428,135, 5,272,064 and 5,149,792 hereby incorporated by reference including drawings); progenitor B cell stimulating factor (see, e.g., U.S. Pat. No. 5,580,754, incorporated herein be reference in its entirety); stem cell factor interleukin-6 (see, e.g., U.S. Pat. No. 5,610,056, hereby incorporated by reference including drawings).

The construct may include sequences encoding proteins engineered such that they will be secreted (see, e.g., U.S. Pat. No. 5,541,083, hereby incorporated by reference including drawings; von Heijne, J. Mol. Biol. 184:99-105 (1985) and references therein, incorporated herein by reference; Rusch & Kendall, Mol. Memb. Biol. 12:295-307 (1995) and references therein, incorporated herein by reference).

In addition, other RNAs, proteins and polypeptides that may be expressed include, but are not limited to insulin; gastrin; prolactin; adrenocorticotropic hormone (ACTH); luteinizing hormone (LH); follicle stimulating hormone (FSH); human chorionic gonadotropin (HCG); motilin; interferons (alpha, beta, gamma); tumor necrosis factor-binding protein (TNF-bp); interleukin-1 receptor antagonist (IL-1ra); brain derived neurotrophic factor (BDNF); glial derived neurotrophic factor (GDNF); neurotrophic factor 3 (NT3); fibroblast growth factors (FGF); neurotrophic growth factor (NGF); bone growth factors such as osteoprotegerin (OPG); insulin-like growth factors (IGFs); macrophage colony stimulating factor (M-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); megakaryocyte derived growth factor (MGDF); keratinocyte growth factor (KGF); thrombopoietin; platelet-derived growth factor (PGDF); colony stimulating growth factors (CSFs); bone morphogenic protein (BMP); superoxide dismutase (SOD); tissue plasminogen activator (TPA); urokinase; streptokinase; kallikrein; interleukin genes; cystic fibrosis transmembrane conductance regulator; human factor VII; interleukin receptor antagonist proteins; cytokines; 4-hydroxyphenylpyruvic acid dioxygenase; tissue-type plasminogen activator or plasminogen; transferlpha-fetoprotein and albumin genes; glucuronosyltransferase; thrombopoietin; immunoregulatory cytokines; phenylalanine hydroxylase; ribozymes; dominant negative genes, and the like; cytochrome genes; immunoglobulin A (Busch, et al., Gastroenterology 115(1):129-38 (1998) incorporated herein by reference); canalicular bile salt transporters; bilirubin uridine diphosphate glucuronosyltransferase; multidrug resistance genes; organic anion transporters; bile salt transporters; ABC transporters, and the like, and other membrane proteins including transporters, receptors, cell adhesion molecules, and the like.

The term protein, as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof. Also included are those proteins with amino acid substitutions which are "conservative" in that they do not affect the intended function of the protein. See generally, Creighton, Proteins, W. H. Freeman and Company, N.Y., (1984) 498 pp. plus index, passim.

Other conditions that may be treated by the compositions and methods of the present invention include inborn errors of metabolism; lymph and blood protein and polypeptide deficiencies or overexpression; cancer (see, e.g., Zhang, W.-W., J. Mol. Med. 74:191-204 (1996)); the several forms of hepatitis and other infectious diseases affecting the liver; hypertension; clotting abnormalities including hemophilia A (Factor XIII deficiency), hemophilia B (Factor IX deficiency) and proteins C and S; obesity; hypercholesterolemia; cystic fibrosis; alpha 1-antitrypsin deficiency; cirrhosis of the liver; diabetes; ornithine transcarbamylase deficiency and other urea cycle related conditions (see Batshaw, Ann. Neurol. 35(2):133-141 (1994)); tryosinemia; phenylketonuria (see Eisensmith & Woo, J. Inher. Metab. Dis. 19:412-423 (1996)); haemochromatosis; carbohydrate disorders, including for example, the several glycogen storage diseases (including glucose-6-phosphatase deficiency); galactosemia and fructose intolerance; lysosomal storage deficiencies; CPS deficiency; FAH deficiency; propionic acidemia; methylmalonic acidemia; defects of branch amino acid degradation (maple syrupe urine disease); lipid metabolism, including LDL receptor deficiency (see Li, et al., J. Clin. Invest. 95:768-773 (1995)), and expression of apoA1 gene for increases in serum HDL levels; porphyrin metabolism by adding porphobilinogen deaminase activity; Wilson disease (copper-transporting ATPase); hyperbilirubemias, including Crigler-Najjar syndrome and Gilbert Syndrome (UDP-glucuronosyltransferase); Dubin-Johnson syndrome (cMOAT); expression of hammerhead ribozymes to cleave hepatitis C virus RNA and inhibit viral protein translation; expressing dominant negative genes, including for example mutants of the hepadnaviral core protein as antiviral agents; correction of Protein C deficiency; correction of Factor X deficiency; expression of antisense RNA, for example anti-sense RNA complementary to hepatitis B virus to inhibit viral proliferation, or to other protein to inhibit its expression; correction of citrullinaemia; expression of p53 to inhibit tumor progression; expression of leptin for obesity; expression of Alpha 1-antitrypsin to treat emphysema; expression of apolipoprotein B mRNA-editing complex to reduce LDL levels; inflammatory bowel disease by expressing immunoregulatory cytokines; attenuation of hypertension and cardiac hypertrophy and renal injury by expression of Kallikrein gene; treatment of tyrosinemia type I; treatment of diabetes by expression of insulin gene, in particular under a glucose responsive promoter/enhancer; treatment of hyperlipidemias associated with apolipoprotein E and LDL receptor deficiencies by expression of human lipoprotein lipase; expression of phenylalanine hydroxylase for phenylketonuria; treatment of hypertriglyceridemia and impaired fat tolerance by expression of lipoprotein lipase; cancer gene therapy; correction of mitochondrial enzyme deficiencies; treatment of mucopolysaccharidosis type VII; secretion of cloned immunoglobulins, including antibodies; treatment of alpha-galactosidase A deficiency and Fabry disease; treatment of dwarfism by expression of growth hormone; treatment of tetrahydrobiopterin deficiency in hyperphenylalaninemic patients by gene transfer of 6-pyruvoyl-tetrahydropterin synthase; correction of diabetic alterations by glucokinase; treatment of type I hyperoxaluria; expression of the enzyme apobec-1 for hypercholesterolemia in LDL receptor-deficiency; treatment of familial hypercholesterolaemia by expression of the VLDL receptor gene; expression of human pyruvate kinase (PK) for gene therapy of human PK deficiency; correction of methylmalonyl-CoA mutase deficiency; treatment of phenylalanine hydroxylase deficiencies; adenosine deaminase and purine nucleoside phosphorylase deficiencies, and the like.

The methods and compositions of the present invention are also applicable for in vivo gene immunization, by direct expression of antigens in the liver, for example.

The present invention also finds applications in animal sciences and for veterinary uses. For example, the present invention is applicable in expressing RNA, proteins and polypeptides in the liver of animals--for action within the liver, or for secretion into the circulation or gastrointestinal tract. The long-term expression, inducible expression, or responsive expression of growth hormone, or growth hormone releasing substances like growth hormone releasing hormone, for example, in adult lactating cows may be achieved without causing the adverse consequences that the constitutive expression of the gene products may have in calves. Similarly, for example, the expression of leptins in farm animals an effective amount of time prior to slaughter for the production of leaner meat, without the adverse consequences such expression would have had had the leptins been expressed in growing and developing animals. The expression of major histocompatibility complex (MHC) molecules, and the like, in cells of the liver of an animal may be altered, for example, by expression of specific MHC molecules, or by inhibition of expression of MHC molecules by anti-sense RNA expression prior to organ transplantation. The methods and compositions may also be used in avian species, for example to produce avian growth hormones, or the like (see eg., U.S. Pat. Nos. 5,151,551 and 5,162,215, incorporated herein by reference in their entirety, including figures). The sequence of the RNAs, polypeptides or proteins encoded by the liver directed gene transfer vector is preferably conspecific.

The combination of factors of the present invention may also be used to enhance the proliferation and transduction of liver cells in vitro.

Claim 1 of 11 Claims

We claim:

1. A pharmaceutical composition comprising tri-iodothyronine (T3) and keratinocyte growth factor (KGF) in a pharmaceutically acceptable carrier, wherein the combination of T3 and KGF is in an amount that induces a semi-synchronous wave of liver cell proliferation upon administration in vivo in a subject.

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