gene therapy for liver disease

The recent discovery of hepatic stem cells andcellular lineages also has great implications to liver gene therapy.. GENERAL PRINCIPLES FOR HEPATIC GENE THERAPY There are two basic approa

CHAPTER 7

Gene Therapy for Liver Disease

CHRISTY L SCHILLING, MARTIN J SCHUSTER, and GEORGE WU, M.D., PH.D.

BACKGROUND

The liver is a complex organ both in anatomy and function These present challenges

as well as provide opportunities for gene therapy of liver disease Anatomically, theliver is a wedged-shaped, mutilobular, large organ In adults, on the average, the livercomprises 1.8 to 3.1% of total body weight In children, the ratio is even larger, up

to 5.6% of body weight at birth The liver receives blood from both the portal veinand the hepatic artery, thus providing systemic ports of entry for therapeuticapproaches The portal vein is the nutrient vessel carrying blood from the entirecapillary system of the digestive tract, spleen, pancreas, and gallbladder The hepaticartery provides an adequate supply of well-oxygenated blood to the liver Innerva-tion of the portal vein and hepatic artery alter the metabolic and hemodynamicfunctions of the liver The functional unit of the liver is the acinus, which is a smallparenchymal mass consisting of an arteriole, portal venule, bile ductule, and lymphvessels A zonal relation exists between the cells of the acini and their blood supply.Different metabolic functions occur in the cells of each zone For example, gluco-neogenesis occurs in cells of zone 1, the area first to be supplied with fresh oxy-genated blood Cells of zone 3 actively metabolize alcohol and biotransform ordetoxify drugs Thus, different zones of liver tissue may need to be targeted fortherapy of metabolic dysfunction The recent discovery of hepatic stem cells andcellular lineages also has great implications to liver gene therapy These discoveriesindicate that cellular characteristics, phenotype, function, and metabolism areunique to a cellular level in the liver as well as based on zonal location Thus, theliver exhibits both microheterogeneity and complexity at various levels that chal-lenge the application of gene therapy to the organ

INTRODUCTION

In the early years of gene therapy, the liver was not taken into consideration as a

153

An Introduction to Molecular Medicine and Gene Therapy Edited by Thomas F Kresina, PhD

Copyright © 2001 by Wiley-Liss, Inc ISBNs: 0-471-39188-3 (Hardback); 0-471-22387-5 (Electronic)

major target organ In contrast to bone marrow and peripheral blood cells, liver cellsare not easily accessible and, in addition, there is no clearly separated pool of liverstem cells Nevertheless, more recently, certain characteristics of the liver havedrawn the attention of many researchers interested in gene therapy The liver hasthe ability to synthesize large amounts of different proteins and performs manyposttranslational modifications required for proper function of those proteins It isalso able to regenerate after partial injury Many systemic inherited disorders such

as hemophilia, familial hypercholesteremia, phenylketonuria, and other metabolicdiseases could be treated by addressing the underlying genetic defect in liver cells

In addition, gene therapeutic strategies could theoretically be used to treat acquireddiseases such as viral infections of the liver Infections by hepatitis B and C virusesare major pulic health problems worldwide For these reasons, the liver has become

an important target organ for gene therapy

At the same time, certain circumstances make the liver an especially challengingtarget for gene therapy The liver is usually quiescent with respect to proliferation,that is, having few dividing cells, and, therefore, not an ideal target for gene vectorsthat require cell division In addition, besides parenchymal hepatocytes, the livercontains a number of other different types of cells These facts should be consideredwhen choosing between different vectors and techniques of delivery of genes to livercells Accordingly, the first part of this chapter will discuss the basic tools, focusing

on their application for hepatic gene delivery, while the second part will address theclinical applications attempted so far

GENERAL PRINCIPLES FOR HEPATIC GENE THERAPY

There are two basic approaches for gene transfer into hepatocytes: ex vivo and invivo strategies (Fig 7.1) Ex vivo therapy requires the removal of a part of the liver

To obtain hepatocytes, the removed tissue is treated with collagenase, and cytes are separated from nonparenchymal cells by density gradient centrifugation.Cells are then kept in culture and subjected to gene transfer by one of a variety ofmethods The population of cells is selected for those successfully genetically engi-neered and finally reinfused via the portal vein into the patient’s liver However,hepatocytes are not readily cultured They undergo a few rounds of cell division butnot enough to substantially expand the population Their viability is limited and cul-turing primary hepatocytes is hampered by some loss of differentiation In addition,

hepato-an already ill patient may not be able to undergo the harvesting procedure.While hepatocytes are kept in culture, several methods can be used to introducenew genes Deoxyribonucleic acid (DNA)-mediated techniques rely on commonlyused transfection methods such as calcium phosphate co-precipitation with DNAand diethlyaminoethyl (DEAE) dextran complexed with DNA through electrosta-tic charges These systems result in complexes that are taken up by the cell via endo-cytosis Electroporation is another technique used to transfect cells This involves theexposure of cells to electrical pulses that render the plasma membrane momentarilypermeable When performed in the presence of DNA, the membrane allows thenucleic acid to enter the cells All three of these methods result in low levels of trans-fection efficiency and transient expression of the therapeutic gene Alternatively, dif-ferent viral vectors as well as liposomes can be used for ex vivo gene transfer

For in vivo gene therapy, the therapeutic or normal gene is introduced directlyinto the host On one hand, in vivo gene therapy circumvents the need for the inva-sive procedures of harvesting and reimplantation and eliminates the need to cultureprimary hepatocytes On the other hand, it is necessary for any vehicle used for in

GENERAL PRINCIPLES FOR HEPATIC GENE THERAPY 155

(a)

(b)

Collagenase treatment and hepatocyte separation

Culture

48hrs.

Reinfusion of genetically altered hepatocytes

Recombinant vector

FIGURE 7.1 Two basic methods for the delivery of genes to the liver (a) Shows the ex

vivo approach It requires the removal of part of the liver, usually the left lateral segment The liver tissue is treated with collagenase and hepatocytes are separated from non- parenchymal cells by density gradient centrifugation Hepatocytes are then propagated in culture and subjected to gene transfer Finally successfully transformed cells are selected and

reinfused via a catheter into the portal circulation of the patient’s liver (b) Shows the in vivo

approach A gene vector, suitable for the delivery of genes to the liver is constructed The therapeutic gene is incorporated into this vector and the recombinant vector is infused into the patient Systemic infusion over a peripheral vein is appropriate for vectors that selec- tively target the liver; direct infusion into the portal circulation is preferrable for vectors without liver targeting abilities.

vivo hepatic gene therapy to reach the liver efficiently For systemic application,the gene vectors are ideally targeted to the liver, avoiding broad biodistribution andextrahepatic effects Once inside the liver, a transgene has to pass through the fen-estrations of endothelial cells to reach parenchymal liver cells, while simultaneouslyavoiding clearance through phagocytosis by Kupffer cells In vivo gene therapy canalso be mechanically directed to the liver by portal injection of the foreign geneconstruct Presently several viral systems as well as liposomal preparations andprotein–DNA conjugates have been used for in vivo gene therapy (Table 7.1).

response Absence of hepatic necrosis Low expression in hepatic

cells in vivo Integrates with stable

expression

specifically Expressed in nondividing Transient expression cells

Inflammatory/immune response

Injurious to hepatocytes Adenoassociated virus Expressed in nondividing Small delivery capacity

cells Integrates with stable expression

No inflammatory/immune response

Large delivery capacity Intracellular degradation in

lysosomes

No inflammatory/immune response

Protein/DNA carriers Liver specific Intracellular degradation in

lysosomes Large delivery capacity Remains episomal

No inflammatory/immune Transient expression response

therapy is that only dividing cells are efficiently transduced To circumvent thisproblem, researchers have performed partial hepatectomies before the administra-tion of the retrovirus Because the remaining liver tissue is induced to proliferate inresponse to this injury, the percentage of transduced cells could be increased.

Adenovirus In early adenoviral constructs, in addition to expression of theforeign gene, some viral genes were also expressed The latter led to a virus-specificimmune response manifested by development of hepatitis and destruction of thegenetically altered hepatocytes The expressed therapeutic protein usually becameundetectable after a maximum period of 4 weeks.The formation of neutralizing anti-bodies by B lymphocytes against viral proteins make a periodic readministrationless effective This problem has been tackled by deleting additional viral genes tominimize the expression of viral proteins It has been shown that the therapeuticgene expression level was increased in mouse liver while the immune response previously seen was decreased Adenoviral constructs have recently been prepared

in which all viral genes have been eliminated Using a different approach, transientadministration of an immunosuppressive drug resulted in the long-term expression

of the adenoviral vector system It has also been shown that it is possible to renderrats immunotolerant to adenoviral antigens by intrathymic injections and oraladministrations of adenoviral protein extracts or by neonatal administration of thevirus in utero, thereby increasing long-term expression and allowing readministra-tion of adenoviral vectors

Adenoassociated Virus Adenoassociated virus (AAV) can infect dividing aswell as nondividing cells making it a possible vector for use in organs such as theliver The rate of transduction in nondividing cells, however, is lower than that ofcells undergoing division AAV transduces cells that are in S phase of the cell cycle.Treatments that interfere with DNA metabolism, such as hydroxyurea or aphidi-colin and topoisomerase inhibitors, markedly increased the number of recom-binant AAV transduced cells.g-Irradiation has a similar effect on the efficiency ofthis system After localized irradiation to the liver, hepatocyte transduction wasincreased up to 900-fold over hepatocytes of mice that were not irradiated This isprobably due to the fact that the irradiation is cytotoxic, thereby stimulating divi-sion of the surviving cells

to the different cell populations within the liver This allows for the targeting

to either hepatocytes or Kupffer cells One advantage of liposomes is the fact that DNA can simply be incorporated in the aqueous phase or associated with the

GENERAL PRINCIPLES FOR HEPATIC GENE THERAPY 157

lipid material In addition, the encapsulated gene is protected from enzymatic degradation.

Cationic liposomes have been used to form DNA complexes in which the DNA remains primarily on the outside of the microsphere While this is an advantage because the DNA that can be trapped within the vesicle is limited, it maycause an aggregation of one or more liposomes and prevent uptake or promote

FIGURE 7.2 Liposomes are used as a device to deliver genes to hepatocytes Liposomes are microscopic vesicles consisting of lipid bilayers enclosing one or multiple aqueous com- partments DNA is incorporated in the aqueous phase or associated with the lipid material after simply mixing with the lipid components Liposomes enter the liver by the portal cir- culation Their clearance from the circulation is largely dependent on their size and surface composition Because the fenestrations of the endothelial cells in the liver have a diameter

of about 100 nm, particles larger than 250 kD cannot pass into the space of Disse Only small liposomes can escape uptake by Kupffer and endothelial cells and interact with parenchymal liver cells.

phagocytosis by Kupffer cells Liposomes are taken up by the cells via endocytosisand eventually enter lysosomes In lysosomes, enzymatic degradation of the contents occurs and could decrease the efficiency of deliver of the therapeutic gene to the nucleus To circumvent this problem, liposomes have been devel-oped that are pH sensitive, avoiding fusion with the lysosomes Following internal-ization, these liposomes change their properties when they are exposed to the low

pH of endosomes During endocytosis, they are able to destabilize the mal membrane or become fusogenic In this way, the liposome may be able to deliver its contents into the cytoplasm before the liposome is delivered to lysosome

endoso-Another means of improving the efficacy of liposomes to target parenchymalliver cells is the incorporation of various ligands recognized by receptors on thesurface of hepatocytes Examples of such targeting moieties are epidermal growthfactor, lactosylceramide, asialofetuin, lactose mono-fatty acid esters, and b-galactoside For many preparations, uptake by endothelial or Kupffer cells com-pared to parenchymal cells is still predominant, and there is no unanimity on thequantitative aspect of the differential uptake into different cell types in the liver.Liposomes with galactose residues are also recognized by Kupffer cells via the galac-tose-particle receptor, and the distribution between parenchymal and non-parenchymal liver cells is strongly size dependent, with only very small liposomeswith limited loading capacity or vesicles containing lactosylceramide or lactosemono-fatty acid esters preferentially directed to parenchymal cells

Protein–DNA Complexes Soluble conjugates between naturally occurring and recombinant proteins and DNA are attractive tools for gene therapy directed

to the liver An example of the use of targeted delivery of protein–DNA plexes is the use of asialoglycoprotein receptors The asialoglycoprotein receptor

com-is present in large numbers only on the plasma membrane of hepatocytes and bindsgalactose-terminated glycoproteins and neoglycoproteins with high affinity Boundligands are internalized by the cell via receptor-mediated endocytosis Due to itsspecificity, the asialoglycoprotein receptor (AsGPr) has been exploited as a means

to deliver drugs and DNA for therapeutic purposes, as well as diagnostic agents

to hepatocytes

A system, based on asialoglycoprotein-poly-l-lysine conjugates has been oped to target DNA to the liver via the AsGPr (Fig 7.3) The a1 acid glycopro-tein, orosomucoid, was desialylated by treatment with neuraminidase to produceasialoorosomucoid (ASOR), a high-affinity ligand for the AsGPr Poly-l-lysine (PL)was then covalently attached to the protein by carbodiimide-mediated amide bondformation The resulting ASOR-PL conjugate bound the negatively charged DNA

devel-in a nondamagdevel-ing electrostatic devel-interaction and protected it from nuclease tion The complex was selectively and rapidly internalized into hepatocytes byreceptor-mediated endocytosis, and foreign genes were expressed in vitro and invivo To further increase the persistence of foreign gene expression in vivo, a partialhepatectomy, leading to stimulated hepatocyte replication was performed Theunderlying mechanism was shown to be the disruption of the microtubular networknecessary for the translocation of endosomes to lysosomes, which could also beaccomplished by colchicine administration

degrada-GENERAL PRINCIPLES FOR HEPATIC GENE THERAPY 159

DNA-negatively charged

Receptor recycling

Hepatocyte carrying the liver specific ASGP-receptor

FIGURE 7.3 Use of asialoglycoprotein (ASGP) to target genes to the liver The coprotein receptor is present in large numbers only on the plasma membrane of hepatocytes and binds galactose-terminated glycoproteins with high affinity Positively charged poly- l-lysine is covalently attached to ASGP by carbodiimide-mediated amide bond formation The resulting ASOR-PL conjugate binds the negatively charged DNA in a nondamaging elec- trostatic interaction The complex is internalized into hepatocytes by receptor-mediated endocytosis After endocytosis the ligand dissociates from the receptor and the receptor recy- cles to the cell surface The translocation of the endosome to the lysosome requires an intact microtubular network After fusion of endosome and lysosome, the DNA is released from its carrier at low pH Part of the DNA escapes the lysosome and reaches the nucleus where it can be transcribed into mRNA.

asialogly-CLINICAL APPLICATIONS OF LIVER-DIRECTED GENE THERAPY

Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is an autosomal dominant disorder that affectsone in every 500 people It is caused by defects in the hepatic low-density lipo-protein (LDL) receptor gene The reduced activity of the LDL receptor leads to

an inefficient clearance of LDL particles by the liver and therefore, a limitedmetabolisim of LDL Accordingly, this causes elevated serum LDL cholesterollevels, which leads to premature coronary artery disease Heterozygotes for FHmaintain only a portion of the normal LDL receptor function, and their serum LDLlevels are almost double that of normal individuals Homozygotes, having twomutant receptor genes, have only 0 to 20% of normal LDL receptor activity andshow extremely elevated serum cholesterol levels Without treatment, this usuallyleads to death by myocardial infarction before the age of 20

The LDL receptor is, in fact, found on all cells However, it is the hepatic sion of the receptor that plays the main role in regulating serum cholesterol levels.The liver is the only organ that is capable of converting cholesterol to bile acids and excreting them from the body Pharmacological therapy for heterozygote FHpatients, who express the LDL receptor at a low level involves upregulation of LDLreceptor gene expression Drugs, including 3-hydroxy-3-methylglutaryl coenzyme Areductase inhibitors and bile acid binders, act to reduce intracellular hepatic freecholesterol This causes the LDL receptor gene expression to be influenced, accel-erating LDL catabolism and, accordingly, reducing serum cholesterol However, thistreatment, combined with strict dietary reduction of cholesterol intake, is only feasible in the case of heterozygosity and does not reduce the serum cholesterollevel into the normal range For those patients that lack expression of a functionalreceptor due to homozygosity, or heterozygotes with an inefficient response to pharmacological therapy, weekly plasmapheresis or liver transplantation are theonly alternatives Both procedures are very expensive, and the latter is associatedwith morbidity and mortality and limited organ supply For these reasons, hepaticgene therapy has been employed in an attempt to treat FH

expres-Early experiments in the Watanabe heritable hyperlipidemic (WHHL) rabbit, ananimal model for FH, demonstrated the possibility of successful ex vivo genetherapy for FH In these studies, hepatocytes were harvested, genetically modified

ex vivo with retroviruses that contained an intact LDL receptor gene, and planted back into the animal Control experiments with mock transfected hepato-cytes demonstrated no cholesterol lowering effect, but showed a transient increase

trans-of the serum cholesterol levels probably due to the surgical procedure Retroviral

transduced hepatocytes were shown to become stably engrafted into the animal’sliver with a subsequent lowered serum cholesterol level The effect was observedfor 6.5 months, the duration of the experiment Subsequent experiments with dogsand baboons also rendered encouraging results In the case of the baboon, 1.5 yearsafter gene therapy, the transgene was still being expressed The results of these earlyexperiments provided support for the efficacy of this treatment and paved the wayfor human clinical trials

A 28-year-old French Canadian woman was the first recipient of liver-directedgene therapy She was homozygous for a mutation in the LDL receptor gene, result-

CLINICAL APPLICATIONS OF LIVER-DIRECTED GENE THERAPY 161

ing in the expression of a nonfunctional receptor After suffering a myocardialinfarction at the age of 16, she had a coronary artery bypass at the age of 26 Herbaseline serum LDL concentration was 482 mg/dl (normal range 194 ± 34), and herdyslipidemia did not respond to conventional drug therapy The left lateral segment

of the patient’s liver, comprising about 15% of total mass, was removed and theparenchymal liver cells were isolated The cells were then transduced with a retro-viral vector containing the full-length human LDL receptor gene under the control

of a chicken b-actin promoter and a cytomegalovirus (CMV) enhancer To select forsuccessful transduction, cells were analyzed for the ability to uptake fluorescentlabeled LDL Only genetically altered hepatocytes were reinfused into the portalcirculation (Fig 7.4) The patient tolerated the procedures well without relevant sideeffects

Immediately following infusion of the genetically altered cells, the patient’sserum LDL dropped by 180 mg/dl A new baseline was established that was 17%lower than before gene therapy As her (LDL) decreased, her high-density lipopro-teins (HDL) levels increased, improving her LDL/HDL ratio from 11 ± 0.4 to 7.9 ±0.9 It is unclear as to why the HDL increased, although this same phenomenon hasbeen observed in patients that underwent orthotopic liver transplantation Thepatient also responded to the drug lovastatin, which prior to gene therapy had

no effect Lovastatin is thought to deplete intracellular cholesterol, thereby regulating expression of the LDL receptor The recombinant receptor gene had notranscriptional elements that could respond to cholesterol-mediated regulation Thisindicates that the response to lovastatin was related to posttranscriptional regula-tion, a mechanism demonstrated in previous studies The response to lovastatindiminished the patient’s serum LDL level further to 356 ± 22 mg/dl, and the effectwas meanwhile stable over a period of 2.5 years

up-There was no immune response to the recombinant receptor The patient’s seracontained no antibodies to the recombinant receptor when a western blot analysiswas performed Also, there was no evidence for autoimmune hepatitis followinggene therapy In an extension of this study, four other FH individuals, including tworeceptor-negative patients, were treated in a similar manner Engraftment of suc-cessfully transduced hepatocytes as well as transgene expression was shown for allpatients, without significant side effects Two out of four patients experienced a significant improvement in their serum lipid profile, with a maximum reduction inserum LDL of 150 mg/dl in one of the receptor-negative patients None of thepatients developed an immune response to the transgene or to retroviral proteins.Although gene transfer was demonstrated in all patients, the clinical impact on thedisease was low with serum cholesterol levels still exceedingly above the normalrange In summary, this first human clinical trial showed the feasibility of ex vivogene therapy for FH but demonstrated the need for substantial modifications toimprove the percentage of transduced hepatocytes and the level and duration ofgene expression

In an alternative approach, in vivo gene delivery was performed to treat WHHLrabbits The human LDL receptor gene was placed under the control of transcrip-tional elements from the mouse albumin gene, conferring efficient expression

in hepatocytes The construct was conjugated via poly-l-lysine to ASOR, a affinity ligand for the ASOR receptor Following systemic injection of this com-plex, analysis of WHHL rabbits revealed a rapid and liver-specific uptake of the

high-CLINICAL APPLICATIONS OF LIVER-DIRECTED GENE THERAPY 163

15%

Left lateral segment

Isolation of hepatocyles Cell culture

Infection with recombinant virus

Surgical removal

of left lateral liver segment

Chromosomal DNA

LDL receptor

Selection by fluorescein labeled LDL uptake

Reinfusion of transduced hepatocyles

enhancer

-actin promoter

LTR Retroviral vector

Transfusion

of genetically engineered hepatocyles

Harvest

of left lateral liver segment

Application

of the reductase inhibitor, lovastatin

HMG-CoA-Serum LDL Cholesterol

FIGURE 7.4 Gene therapy for LDL receptor deficiency The left lateral liver segment of a patient homozygous for a mutation in the LDL receptor gene is removed and hepatocytes are isolated The cells are transduced in culture with a retroviral vector containing the full- length human LDL receptor gene under the control of a chicken b-actin promoter and an cytomegalovirus (CMV) enhancer The successfully transduced cells are selected by the use

of fluorescein-labeled LDL Only genetically altered hepatocytes are reinfused into the portal circulation of the patient The patients baseline serum LDL concentration was 482 mg/dl Immediately following infusion of the transduced hepatocytes, the patients serum LDL dropped by 180 mg/dl In addition the patient now responded to lovastatin, a HMG-CoA reductase inhibitor, which prior to gene therapy had no effect The observed reduction in the patients serum LDL level is meanwhile stable over a period of 2.5 years.

DNA–protein conjugate, followed by expression of the transgene The animals experienced an immediate, but transient, decrease in total serum cholesterol by

153 ± 53 mg/dl In control experiments, animal injected with a construct carrying theCAT (chloramphenicol acetyltransferase) reporter gene instead of LDL receptorgene showed CAT expression, but no diminuation of serum cholesterol levels Inthis study the expression was only 2 to 4% of the endogenous level of LDL recep-tor expression, and the effect on the serum lipid profile lasted less than one week.These initial results were encouraging because of the specificity of the delivery.However, the low levels and short duration of recombinant gene expression weredisappointing

In recent animal studies, recombinant adenoviruses were used for in vivo directed transfer of the LDL receptor gene It was possible to restore LDL recep-tor expression in WHHL rabbits and LDL receptor knock-out mice, leading tosubstantial reductions in serum cholesterol levels However, the expression of therecombinant receptor as well as the effect on the lipid profile has been only tran-sient This was due to the immune response that the host mounted against a low-level expression of viral proteins, with the subsequent destruction of the geneticallyaltered cells Especially in receptor-negative subjects, the expression of an LDLreceptor could also trigger an immune response against the neoprotein, which wouldfurther reduce the expression of the transgene To circumvent this problem, anothergroup of researchers delivered the very low density lipoprotein (VLDL) receptorgene to the liver of LDL receptor knock-out mice using recombinant adenoviruses.Since the VLDL receptor is already expressed in extrahepatic tissue, there is noimmune response to the receptor after hepatic expression Also the VLDL recep-tor binds LDL with a low affinity It mediates the uptake of VLDL, the precursor

liver-of LDL, and, therefore, results in a decrease liver-of serum cholesterol

Hemophilia B

Hemophilia B is an X-linked recessive coagulation disorder caused by a deficiency

or functional defect of blood clotting factor IX The condition can be life ing without regular infusions of factor IX concentrates in patients with evidence ofbleeding Extensive testing of these products can eliminate impurities, but this form

threaten-of therapy still bears the risk threaten-of transfusion-transmitted viruses such as hepatitis Cand human immunodeficiency virus (HIV) In addition, the half life of factor IX isonly 24 h and, therefore, makes repeated transfusions often necessary The liver isthe primary source for circulating factor IX and the prime target for a gene thera-peutic approach to treat hemophilia B

To date, attempts have been made in animal systems using the ex vivo approach.The problems with these therapies are similar to those that have been encounteredwith correcting other disorders: (1) the concentrations of circulating factor IX arelow and (2) there is a loss of gene expression over time The latter is due to loss oftransduced cells or inactivation of the expression vectors

There is a well-characterized canine model that has been used in preclinical trialsfor hemophilia B These dogs have no detectable factor IX activity due to a mis-sense mutation in the catalytic domain.A retrovirus vector that contained the caninefactor IX gene under the control of retroviral promoter and enhancer elements wasused for direct delivery to the dogs liver via infusion into the portal circulation

Analysis by ELISA and a biological assay demonstrated that plasma levels of 2 to

10 ng/ml of factor IX were achieved In a normal canine, the level is about 11.5mg/ml.While the levels of circulating factor IX did not reach that of wild-type dogs, therewas a dramatic improvement in the biochemical parameters of hemostasis This wasdemonstrated by measuring the whole blood clotting time (WBCT), which innormal dogs is 6 to 8 min In dogs that have hemophilia B, the WBCT was about 45

to 50 min After undergoing gene therapy this time was reduced more than 50%with times in the range of 18 to 22 min Although the concentration of factor IX was

as little as 0.1% of normal values, there was a dramatic improvement in clottingtimes Also, encouraging is the fact that this effect remained stable for over 9 months(Fig 7.5)

Adenoviral vectors that express canine factor IX have also been used to treathemophilia B dogs Viral particles (2.4 ¥ 1012) were infused into the portal vasculature of the dogs The animals produced 2 to 3 times the wild-type level offactor IX However, the effect was only transient The increase in factor IX con-centration did normalize their clotting times, but the levels and clinical parametersreturned to pretreatment levels in 2 months While repeated administrations could

be considered, it is possible that an immune response could develop with quent treatment

subse-Another group of researchers tried using adenoassociated viral (AAV) vectors

to express human factor IX in mouse livers They simply injected the mice in a tailvein with the recombinant vector after g-irradiation was applied to the liver As pre-viously discussed, this treatment probably stimulates cells to divide, thereby improv-ing the efficacy of adenoassociated viral gene therapy The concentration of humanfactor IX in mice transduced with the AAV vector was between 0.1 and 1 ng/ml Thisresult is similar to that observed in the dog model The normal values for humanfactor IX was 5mg/ml, while levels of about 100 ng/ml would prevent chronic disease

a1 -Antitrypsin Deficiency

a1-Antitrypsin (AAT) is a serum glycoprotein, predominantly synthesized in theliver and secreted into the blood It is a protease inhibitor whose function is essential in protecting the alveolar surface of the lung from destructive proteaseactivity Its major substrate, neutrophil elastase (NE), is released by neutrophilsduring phagocytosis, membrane perturbation, or cell lysis and cleaves connectivetissue matrix proteins located in alveolar walls In normal individuals the levels

of AAT are sufficient to neutralize circulating NE The different forms of AAT deficiency result in reduced plasma levels of the protease inhibitor and in the failure

of NE to be neutralized This is manifested in a high risk for the early ment of pulmonary emphysema, due to proteolysis of the pulmonary extracellular matrix

develop-The normal gene for AAT is designated M and accounts for 95% of alleles in thecaucasian American population The most common mutants, called Z and S occurwith an allelic frequency of 1 to 2% and 2 to 4%, respectively, in this population Incontrast Asians and African Americans are minimally affected Homozygous indi-viduals for the Z allele have only 10 to 15% circulating AAT levels bearing a certainrisk for pulmonary emphysema Homozygous individuals for the S allele and MS or

MZ heterozygotes are phenotypically normal However, some SZ heterozygotes

CLINICAL APPLICATIONS OF LIVER-DIRECTED GENE THERAPY 165

could display an increased risk for the manifestation of pulmonary emphysema.

Homozygosity for the so-called null allele results in a complete lack of AAT in theplasma, and these patients are extremely likely to develop emphysema The same

is true for heterozygotes bearing an S or Z allele with the null allele A number of

Retrovirus carrying

the factor IX gene

Infusion into factor IX deficient dog via portal circulation

Infection of Hepatocytes

and chromosomal integration

of the Factor IX gene

Test for gene expression

and clinical effects

Treated Dog Normal Dog

Clotting Time (min) 50

10

Retrovirus injection

Factor IX deficient dog

Normal Dog

Treated Dog Factor IX Expression

FIGURE 7.5 Gene therapy for factor IX deficiency A recombinant retrovirus vector is structed that contains the canine factor IX gene under the control of retroviral promoter and enhancer elements (LTR) This vector is infused into the portal circulation of dogs that have

con-no detectable factor IX activity The retrovirus is taken up by liver cells and the provirus DNA integrates into the chromosomal DNA Analysis of the dogs’ plasma by ELISA reveals plasma factor IX levels of 2 to 10 ng/ml A normal canine has a plasma factor IX level of about 11.5mg/ml While the levels of circulating factor IX in the treated dog does not reach that of wild-type dogs, there was a dramatic improvement in the whole blood clotting time.