BIOLOGY

OF REPRODUCTION

42, 39-49

(1990)

Prospects

for Gene Therapy

EVELYN Laboratory National

M. KARSON’

of Molecular Heart,

National Building

Hematology

Lung, and Blood institute Institutes of Health 10, Room 7D-18

Bethesda, Maryland

20892

ABSTRACT Experiments in animal be useful to treat genetic

models and hwnan cells in vitro suggest that gene transfer using retroviral vectors may diseases and to gain information that may improve treatment of other common diseases such as cancer. The approach to treatment of genetic diseases fry inserting genes into bone marrow cells and experimental models, and a novel application of gene transfer technology to cancer research are discussed herein.

transferase (NPT), that inactivates several antibiotics, including one called 0418. G418 is toxic to mammalian cells, so that one can select for cells containing the inserted gene and against cells that do not contain the NeaR gene. The vector N2 contains the NeoR gene promoted by the regulatory sequences LTR (long-terminal repeat) region, whereas the SAX vector, in addition, has the gene for human ADA under the control of the early promotor of the SV4O virus.

INTRODUCTION The purpose of this paper is to present an overview of research that utilizes gene transfer to treat human diseases. First, some of the principles of gene transfer using retroviral vectors will be introduced. The focus of the remainder of the paper is the potential of this technology for treatment of disease. The following topics will be addressed: 1) which genetic diseases are approachable in the near future, 2) the biological crite-

ria for choosing these diseases, 3) what types of cells might be treatable, 4) experience with animal models, and 5) a novel application of gene transfer technology to improve cancer research. RETROVIRAL GENE

VECTORS AS SYSTEM

WHICH

There are several important considerations, both ethical and scientific, in choosing the initial candidates for gene therapy. When introducing a new, unproven technology, the risks are not completely quantifiable. Consequently, only severe disorders, those for which current theory cannot stop the progression to severe crippling or early death, should be considered for treatment initially. Scientifically, it is important to begin with the fewest complicating biological variables in order to maximize the likelihood of success in the initial trial. Specific biological requirements would include the following: 1) A recessive disease caused by the absence of a normally functioning copy of a gene. Treatment of autosomal dominant diseases, in which the presence of one abnormal copy of a gene causes disease, would remain unapproachable until methods for “turning oft” or removing defective genes have been perfected. 2) The gene has been cloned.

A

DEUVERY

A retroviral vector is a defective recombinant retrovirus in which all or part of the three structural genes, gag, pol, and env, necessary for viral replication, have been replaced by the gene(s) of interest This particle retains the ability to infect and integrate into a single target cell and deliver its genetic information, but not to replicate. Examples of the structure of the Moloney murine leukemia virus and two derivative retroviral vectors, used in the experiments to be described below, are shown in Figure 1. Each contains a gene called Neo, which produces an enzyme, neomycin phospho-

‘Reprint

DISEASES ARE CANDIDATES FOR TREATMENT?

requests.

39

40

KARSON

3) The gene in the normal person codes for a single polypeptide that does not need to be located in a highly specialized cellular environment to function effectively. 4) The normal function of the cell does not require precise regulation of the amount of the protein produced. 5) The disease can be ameliorated or corrected by treatment of a target cell that is directly affected by the disease process or another cell type that can produce new gene product. In the latter case, either the gene product can function at a distance from affected tissues, for example, to lower the concentration of a toxic byproduct or to increase a necessary metabolite, or the target cell can produce and deliver the gene product to affected organs. 6) The target cells for treatment can be safely removed from the body for manipulation and then returned.

7) Although not mandatory, it is desirable that the treated cells have a selective growth or survival advantage so that elimination of the patient’s remaining untreated cells would not be necessary.

been shown to transfer lysosomal enzymes directly to fibroblasts (Olsen et al., 1981; Abraham et al., 1985). Unless the gene product requires cell-specific posttranslational modifications or specific localization in the true target cells affected by a particular disease, the transfer of exogenous genes with appropriate regulatory signals may allow bone marrow-derived cells to produce proteins not usually expressed. For instance, phenylalanine hydroxylase, the enzyme that is not functioning normally in many cases of phenylketonuria, is not ordinarily synthesized by blood cells. The cloned gene inserted into cultured cells has been shown to produce active protein (Ledley et al., 1986). Because dietary restriction decreases intellectual impairment, suggesting that the central nervous system (CNS) damage in phenylketonuria is secondary to accumulation of phenylalanine or a toxic byproduct rather than cerebral deficiency of tyrosine, it is possible that creating a peripheral blood pool of enzymatic activity may be effective treatment without more specific targeting of the enzyme. Other

Bone Marrow

as a Target

Organ

Based on the success of bone marrow transplantation (BMT) in treating a variety of genetic diseases, prospects for the use of bone marrow as a target for gene transfer have been reviewed by Parkman (1986). Some of the treated cells, the totipotent stem cells, after having been removed from the body for processing and then reinfused, may have the capacity for self-renewal throughout the lifetime of the patient. A large number of cells from other organs, many of which cannot propagate and currently cannot be easily transplanted, interact with the many types of blood cells and macrophages that differentiate from the bone marrow cells. Thus, many disorders besides those directly affecting cells derived from bone marrow may be correctable through use of genetically engineered bone marrow. For example, the enzymes affected in lysosomestorage diseases have a characteristic that makes them particularly suitable for indirect delivery via gene therapy using BMT; post-translational modifications add a mannosyl-phosphate carbohydrate side-chain that targets their uptake into lysosomes. Liver biopsies obtained from a patient with Hurler syndrome after BMT demonstrated that enzyme from donor Kupifer cells (which are a type of bone-marrow-derived macrophages) cleared hepatocytes of mucopolysaccharide (MPS) inclusions. Lymphoid cells in vitro have also

Possible

Target

Cells

Preliminary in vitro experiments with human cord blood obtained at the time of premature and term deliveries and experiments with fetal sheep and nonhuman primates, (discussed in a later section) suggest that it may be feasible to use circulating hematopoietic progenitor cells as an alternative source of “bone marrow” cells. During the hematopoietic expansion in the fetus, these cells migrate from the liver and spleen to the bone marrow, which becomes the major site of hematopoiesis in later life. The treatable cell pool in the fetus can be removed by percutaneous umbilical vessel blood sampling under ultrasound guidance (PUBS), transduced in the laboratory, and reinfused by PUBS or ultrasound guided intraperitoneal injection. This method would be particularly useful in treating diseases where there is significant deterioration by the time of birth and might possibly permit cells assess across the bloodbrain bather. Alternatively, where these factors are less important, there may be an advantage to waiting until after birth for treatment. One could aseptically collect fetal cord blood at delivery for treatment and reinfuse the cells by a partial exchange transfusion, if necessary. A larger fraction of the blood can be sampled at this time, and the risk of the actual manipulations to the mother and child are reduced. In either case, repopulation of the hematopoietic system with the “repaired” fetal cells, particularly in cases where these cells have a

GENE PP II

SK

I

MoMuLV

X I

S I

THERAPY C

K I

1 LTR

gag

5’q

PP

E

P

PXEC

II

II

I

I

III(

LTR

SK II

I LTR

3’

p0/

SK

N21

I

5’ q

SK

NEOR

LTR

PP

E

p

p

II

I

I

I

pPECSK

1

SAXI LTR

NEOR

5’’I’

SV4O

hADA

LTR

I

I

I

I

I

I

I

I

I

I

I

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

SCALE FIG. 1. Structures of a relrovirus vectors were constructed. early prosnolor. retroviral

41

and reuoviral N2 is a shuttle

5.5

6.0

6.5

I

I

I

I

I

7.0

7.5

8.0

8.5

9.0

IN Kb

vectors N2 and SAX. MoMuL V. Moloney murine (mouse) leukemia virus is the retrovirus from the Nec gene and SAX is a shuttle for both NEOR and human adenosme deaminase (hADA)

for

selective advantage for survival, might be accomplished without the risks associated with radiologic or chemotherapeutic ablation that are usually necessary to achieve engraftment in later postnatal life. By using gene transfer, other types of tissues may also be used as shuttles or generators of the normal gene product for diseased patients. Because of their contiguity with the bloodstream, endothelial cells are a particularly attractive target that would be suitable for enzymes and proteins normally found in the blood as well as those that can function there as an alternative (such as the examples described above). Endothelial cells could be isolated from the patient, expanded and treated cx viva, and then be returned to the patient either to directly seed the blood vessel or in the form of an artificial graft or shunt. Expression of exogenous genes in cultured rabbit endothelial cells before and after seeding onto synthetic vessel grafts have been shown in vitro (Zwiebel et al., 1989). Fibroblasts from an individual can also be propagated in vitro and transduced with exogenous genes. Cells can be immobilized on collagen matrices and be reimplanted to provide a source of enzyme that either migrates to an affected tissue or that can function autonomously. Application of an angiogenesis factor to the matrix can promote vascularization which will sup-

port survival 1988). initial

of the implanted

cells

which with

(Thompson

the two the

SV4O

et al.,

Candidates

In the past, clinical investigators thought that the human genetic diseases most likely to be the initial candidates for successful treatment by gene therapy would be the hemoglobinopathies such as beta-thalassemia. Bone marrow is one of the easiest tissues to manipulate outside of the body, but hemoglobin is composed of two subunits coded by genes on two different chromosomes and switches from embryonic to fetal to adult forms. Moreover, the levels of each subunit are controlled by complicated regulatory signals. Currently, the form of severe combined immunodeficiency disease resulting from adenosine deaminase (ADA) deficiency is likely to be the first to be treated by gene therapy. Children with ADA deficiency have recurrent infections of the skin, CNS, and sinopulmonary and gastrointestinal tracts, as well as failure to thrive because of chronic diarrhea and malabsorption, and a high incidence of lymphoma. Most individuals who do not have a histocompatible bone marrow donor die in early childhood. Because BMT can be curative, and the other criteria described above are met, it is

KARSON

42 thought marrow normal therapy.

Other

that an “autologous” transplantation of bone cells that have received ex vivo a copy of the ADA gene could be the first successful gene

Potential

Candidates

From a theoretical perspective, what other categories and specific diseases might be candidates in the near future? Table 1 lists some of the diseases in which the gene of interest has been cloned and for which there are varying possibilities of successful treatment in the near future. Subset A includes several of the examples given and other similar diseases for which gene therapy holds much promise. Subset B gives examples wherein the affected gene codes for blood cell structural protein in which the addition of normal molecules to these cells is likely to ameliorate the abnormal morphology, despite the continued production of abnormal protein (in contrast to the problem of cirrhosis in a1-antitiypsin deficiency discussed below). In subset C, the problems of complex regulation may complicate the application of gene therapy for some time.

Unlikely

Candidates

Regrettably, structural proteins for most tissues such as muscle, visceral organs, or neurons probably cannot be supplied via BMT (Table 2). The gene for a1antitrypsin has been cloned and expression of the functional, glycosylaled protein in fibroblasts in culture has been achieved (Garver et al., 1987), but only partial correction of the clinical defects may be possible. Theoretically, production of the protein by the stem cell-

TABLE

1. Prospects

for gene therapy:

diseases

A. Likely to succeed: Menosine deaminase deficiency Argininiosuccinic aciduria (argininosuccinase) Ciuullenemia (argmosuccinale syntbetase) Gaucher disease type I (glucocerebrosidase) Phenylketonuria (phenylalanine hydroxylase) Purine nucleoside phosphorylaae deficiency B. Possibility of success Elliptocytosis 1 (protein 4.1) Elliptocytosis 2 (spccrnn) Granulocyte actin dcficiy C. Cosnplex regulation Coagulation factors VIII, IX, X, XJIla Cosnplement factors C2. C4, C9 Hnogithinopwlacs Hereditary angioneurolic edema (Cl inhibitor) l’halassemia

with cloned

genes.

TABLE targeting

2. Diseases problem.

unlikely

to be cured

by gene therapy

u1-antitrypsin deficiency Carbamyl phosphate synthetase deficiency Fatay disease (alpha galactosidase) Fucosidosis (alpha fucosidase) Gaucher disease types II and HI Hypophosphalasia (alkaline phosphatase) Metachromatic leukodystrophy-.variant (SAP Lesch-Nyhan disease (HPRT) Grniihine transcarbamylase deficiency Propionyl CoA carboxylase deficiency Sandhoff disease (hexosaminidase A and B) Tay-Sachs disease (hexosaminidase A)

in the near future:

1)

derived alveolar macrophage (Thomas et al., 1976) may be a useful treatment for the emphysema component of the deficiency disease, but the cirrhosis that is postulated as resulting from the deposition of nonsecreted protein will progress. Indeed, lransgenic mice in which the human PiZ mutant protein is expressed show liver histopathology similar to homozygous affected patients (DeMayo et al., 1986). At present, it is not certain whether bone marrowmediated gene transfer will be applicable to diseases with significant manifestations in relatively inaccessible body spaces such as bone matrix or the CNS. CNS manifestations of disease can arise by a variety of mechanisms, only some of which may be correctable with currently anticipated technologies. Damage to the brain and/or neurons may result from physical compression, either intraceilularly by direct accumulation within the cell organelles or cytoplasm, or extracellularly from adjacent tissues on either side of the blood-brain barner. Alternatively, accumulation of toxic metabolites or absence of a necessary substrate may cause deterioration or developmental failure that may or may not be reversible. CNS problems originating outside of the blood-brain barrier are more likely to be reversible with treatment In addition, microglial cells are hypothesized to arise from bone marrow-derived cells that are capable of crossing the blood-brain barrier (Konigsmark and Sidman, 1953; Bartlett, 1972). It is not clear, however, if engrafiment in the brain can be achieved without using harsh ablative treatment. One reason for considering treatment of the fetus or the newborn is that the blood-brain bather may be more permeable to treated cells in these younger patients. EXPERIMENTAL TRANSFER

PROTOCOLS FOR GENE IN ANIMAL MODELS

Several of the experimental protocols vectors from the laboratory of W. French

using these Anderson at

GENE TABLE

3. Primate

BMT/gene

transfer:

summary

for hADA

gene. Activity

THERAPY

43

of the NeoR and the human

ADA genes

measured.

were

Enry No.

Name

1 2 3 4

Bill (C)d Mork(R) Mindy (R) Kate(C) Ethel (K) Robert (C) Kyle (C) Venus (R) George (C) Ken (C) Oppie(C) Barney (C)

5

6 7 S 9 10 11

12

Date 7/12/85 9,06/85

9106185 10129/85 10129/85 11/19/85 11/19185 11120/85 3/19/86 3/19/86 8,06186 8106/86

‘Transduction using either the cocultivation (C) or supernatant = neophosphospholransferase activity. C%

Endog

=

amount

#{188}C) cynomolgus =

of human (R)

=

ADA activity/endogenous

Method’

Reconstitution

C C C C S S S C S S S S

No No No No Yes Yes Yes No Yes Yes No Yes

(S) method

monkey

activity

in blood ADA-%

Pos