Gene transfer for erythropoiesis enhancement

Senior Scientific Director, omatix Therapy Corporation, y, Alameda, CA 94501, USA.

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ofthesepmgmitorsinto anemia that results from in which the kidney cells that produce the highest quantities of thi hormone are destroyed. It is also administered to patients with rheumatoid be associated with anemia. lation of about 8 h, and long injections of SO-500 units therapy has been proposed for a variety of short-term applications, including anemias caused by chemotherapy or associated wi birth (Table 1). PII: S 1357-43 lo(96) 10033-2

3

(Table 1) (for a review on the use of current therapeutic regimes using volve frequent hospital visits. The treatment of large numbers of patient: chronic renal failure or the use of doses of Epo for patients with hemoglo-

.

/-

BFUe

idney: peritubular cells Liver: hepatocytes and lto cells Brain: astrocytes Synthesis of _O HIF-1 @to

Transcription of gene for EPo

-

Epo

CFUe

effect of Epo secretion can be easily monitored over time, in experimental animals and in humans, by measuring the crit. Thus, beyond its clinical re gene transfer of Epo has becom able paradigm in which to study the in Go delivery of therapeutic proteins, because of the convenience of measuring t effects of this hormone irz Y&X

precursors A

Erythrocytes

4

Vi

s

One strategy for delivery of Figure 1. Erythropoietin (Epo).mediated regulation of erythropoiesis in response to oxygen demand. The engineer cells e.y U~IYIin order to make amount of oxygen delivered to the tissues is controlled by the number of erythrocytes in the bloodstream. them produce Epo, and to graft the modiEpo is involved in the feedback circuit that adjusts the rate of erythrocyte production when tissue oxygenfied cells as an autologous implant to proation is either increased or decreased compared with the normal level. Blood loss or decreased atmospherip vide the recipient with the hormone. oxygen at high altitude results in decreased oxygen delivery to the tissues. Epo synthesis, which occurs one marrow is the tissue most amenable mainly in kidney peritubular cells, is then stimulated in response to signals from a hypoxia sensor, probably a heme protein. A nuclear factor induced by hypoxia via de nova protein synthesis, designated hypoxiato this type of manipulation: the modifiinducible factor 1 (HIF-I), activates transcription of the gene for Epo. In the bone marrow, Epo binds to a cation of stem cells in the marrow would specific ceil-surface receptor on erythroid progenitor cells, which is expressed mainly on colony-forming-unit offer a means to create a permanent, selferythroid cells (CFUe), although burst-forming-unit erythroid cells (BFUe) and erythroid precursors also renewable implant. Although the technolexpress the receptor. Binding of Epo to this receptor promotes the survival, proliferation and differentiation of these progenitors into erythrocytes. Conversety, when tissue oxygenation is increased above normal, plasma ogy is still inefficient in large mammals Epo levels decrease and erythrocyte production is reduced, which results in decreased oxygen delivery to and humans, this strategy has already the tissues. proved effective in mice5. Using a retroviral vector, Villeval et al. have transferred the monkey Epo cDNA into murine __ Another application comes from the notion that synthesis of y-globin bone marrow cells; these cells were then transplanted into syngeneic in erythropoietic cells is stimulated in the presence of Epo. This pro- recipient mice. Following transplantation of the modified hemopoietic vides a rationale for the treatment of those hemoglobinopathies in cells, the mice showed elevated monkey Epo levels in the serum and which P-globin chain synthesis is affected, such as P-thalassemia or high hematocrit value?. The same protocol was applied to homozysickle cell anemia. Although the y-globin does not significantly par- gous P-thalassemic mice, which carry a deletion of the gene for ticipate in the synthesis of adult hemoglobin (HbA, a,Q,), its producP-major globin and have clinical and biological features similar to tion could compensate for a P-globin defect by the production of those observed in human P-thalassemia’. In these animals, the transfetal hemoglobin (HbF, cxzy2).A therapeutic effect of repeated injecplantation of bone marrow cells engineered to secrete monkey Epo tions of rHuEpo in association with the drug hydroxyurea has been had the same effect as repeated injections of rHuEpo: expression demonstrated in patients with sickle cell disease. Treated patients show of the gene encoding p-minor globin (the mouse equivalent of the increased HbF synthesis, decreased polymerization of abnormal sickle human y-globin chain) was stimulated, leading to an improved hemoglobin (HbS) and improvement of the rheologic properties of erythrocyte phenotype and an elevated hematocrit. This study indierythrocytes. rHuEpo has also been administered to patients with cated that Epo gene therapy was feasible. However, a safety concern thalassemia intermedia, and their anemia was improved. However, the was also raised as a lethal polycythemia (abnormally high production treatment of all of these hemoglobinopathies required doses about of erythrocytes) was induced in several of the treated mice: using this tenfold higher than required for the treatment of acquired anemias strategy it was not possible to modulate the levels of Epo. I

344

ent Lifelong, or until kidney cell function restored by renal tran~lantatio~

Chronic renal failure B_Thalassemia and sickle celi disease nt

lunl-t Rheumatoid arthritis Cancer AIDS

8

150-200

M-200 100-500

nt

Cancer: anemia following chemotherapy or radiation therapy Prematurity (infants)

Several momths Seweral months Several months Up to three months Up to one month

1 S - cek that Can WEbW differentiate into every type of myeloid present at low levels in the bone marro gene transfer into the hematopoietic system genetically modified bone marrow stem cells perpetuating cell mass that contains t tire lifespan of the patient.

0

8

Weeks

i3t-d

40

18

after implantation

t~emSe~V@S

of neo-organ

a discrete area where the cells have The quantification of these plaques g

s - In order to transfer a mutati a large number of backcross m

are necessary. The resultkg animals are syngeneic. Cell pk.;;:ation frcllln one of these animals to another does not result in immunological rejection. of this review, a means of Some vectors are derived viral vectors include DNA-lipid and DNA-protein complexes. 0 8

18

40

Weeks after implantation Figure 2. (a)The effect of etythropoietin (Epo)-secreting neo organs on the hematocrit of mice. Six-week-old DBA/2J mice were implanted intraperitoneally with 2 x 10’fibroblasts transduced with a retroviral vector containing the murine Epo cDNA under the transcriptional control of the cellular phosphoglycerate kinase promoter. Peripheral blood was collected retroorbitally into heparinized microtubes and centrifuged to monitor the hematocrii every seven days. Three out of six mice were followed up to ten months after implantation. In untreated animals, the hematocrit remained between 42% and 47% (shaded area on graph). (b) Plasma Epo levels. At T,, (before implantation), T, and T2 (day 56 and day 126 after implantation, respectively), plasma Epo levels were measured by enzyme-linked immunosorbent assay and ranged from 60 to 408 milliunits ml-’ (the means were 189 k102 milliunits ml-’ at T, and 275.2 * 118.2 milliunits ml-’ at T,) and were therefore significantlyhigher than preimplantationvalues (~20 milliunitsmlS1; P < 0.001). In untreated mice, Epo plasma levels were unchanged.

of genetically modified cells’6.Accordingly,long-ten Epo delivery following injection of a recombinant adenovirus has been mostly observed in immunocompromisedadult animals or in neonates in whichthe immuneresponseto the recombinantvirus is normallydepre~sed*~ (Fig.3). A transientEpo production,lastingonly a few weeks, was documented in intravenouslyinjected miceI and in cotton rats receiving a single i.p. or subcutaneousdose of recombinantadenoV~IXS carrying the human gene for Epo”. Nevertheless, long-term Epo production, with a hematocritreaching 80%, was demonstrated 346

cular (i.m.) in immunocompetentmice fo for murine injection of recombinant aden Epo”. This study suggests that the immune response directed against the foreign (human)Epo is the primaiy cause of the disappearanceof gene expression in rodents”. In addition, Descamps et al. have reported stable expression of the gene for monkey Epo for six months following a single intravenous injection with high doses of a recombinant adenovirus in adult DBA/2J mice2’.Although these data are seeminglycontradictory,they indicatethat the longevity of expression is a function of the intensity of the immuneresponse, and that it will vary dependingon the antigenicityof the transgeneproductand on the geneticbackgroundof the experimentalanimals2’Transient . production of Epo could be clinically relevant for short-termhormone treatment needed by patientsreceiving chemotherapyor radiotherapy.Tripathy et al. have estimatedthat successful clinical treatment of the anemia related to chronic renal failure would require a single i.m. injection of 3.5 X IO9plaque-formingunits of recombinantadenovirus”. e for

On the whole,publishedstudies demonstratethat sustainedin vivo delivery of recombinantEpo can be achieved using a variety of gene transfer protocols.Before clinical applicationscan be further considered, the stability,tissue specificity and re.gulationof Epo expression mustbe carefullyinvestigated.As discussedabove,most clinicalapplications would require long-term expression of a transgene for Epo. Stability of expressionwill be determinedboth by the persistenceof

transgene

expressisn to be comtro%%ed: ~e~~~~~~a~vecm-s offer the

vector doesnot exist yet. ial for pe ent rnodifirect id ~3 pplications nes to non-dividing cells. -codinp sequences from enovirail vectors as these will stinnuiahe an immune response that minates transduced cells. The potential of adeno-associated-virus axtors and ~~~-~l~~a~ are investigated irr \*i\~. and ihe gene for Epo should ter m these mdies as it5 etieces irl \,i\.o are easily monitored. The current

or worsening of renal failure caused by acceleration of l~ypertensiorited glomeruhr injury’h. In gene tn’anlsferexperiments in animal s, a simple dose-response re~a~~oi~sb~~ was observed between serum levels of Epo and either the amount of recombinant adenovirus injected or ths number of engineered fibroblasts implanted into neoorgans’.“. This suggests that straightfor the administered doses might be possib vectors, treatment could be inittated wi optimum dose could be determined implies multiple administrations of the recombinant virus, which cannot be achieved with the current adenoviral vectors”. With the neoorgan approach, implants of genetically modified cells could be added or removed if necessary, but this procedure might be too traumatic or too complicated to be practicable. An ideal way to achieve secretion of a regulated amount of Epo might be by using the natural control elements of the human gene for Epo. Hypoxia regulates Epo synthesis in the kidney primarily through the rate of gene transcription; hypoxia-responsive cis elements have been localized to the 3’region of the gene for Epo and their size is suitable for insertion into any existing viral vecto?‘. These cis elements have already been combined with the adenovirus major-late promoter in a recombinant adenovirus carrying the gene for human Epo”. ltt rift-u studies showed that, following infectian with this vector, the production of Epo from the human hepatoccte celi line Hep3B was enhanced 1IQ-fold following a hypox.,ir stimulus. Of potential interest is the fact that the oxygen-sensing system initially identified in Epo-producing kidney cells and (to a lesser extent) liver cells has now been found in a wide variety of cell types”. However, recent data from transgemc mice containing the gene for Epo with various combinations of flanking sequences and introns suggest that the precise regulation of basal and

Control

I

1

I

I

I

I

Time (days) Figure 3. Monitoring of the hematocrit in mice Injected with a recombinant adenowins. Eight-week-old severe combined immunodeficrent mice (n = 3-8) were Injected once intramuscularly with i O’-10’ plaque-forming units (pfu) of a replication-defective adenovirus containing the cDNA for human Epo under the transcriptional control of ;‘:e cellular elongation factor 101 promoter and the 4F2 heavychain enhancer (red lines). Control animals were injected with 10’ pfu of an adenovirus carrying the /acZ gene (blue tine). Hematocnts were measured by centrifugation of blood obtained from tail veins at the times in&ated. Modified from Ref. 17.

/

Control of expression of the gene for Epo is necessary to avoid overproductionand to allow doses to be varied. This might be achieved by using the &-acting regulatory elements from the gene for human Epo itself; however, the current vectors might not be able to accommodate the numerous elements that could be required. and these elements might turn out to function differently depending on the cell type. Heterologous systems, such as tetracycline-regulable g3romoters, are candidates for in viw regulation of transferred genes, and again the Epo system is convenient to test their efficiency’“. It is likely that when vector technology has advanced sufficiently, an optimal combination of vector and gene-transfer procedure will be defined for each situation in which erythropoiesis enhancement is sought, For clinical applications requiring only two or three weeks of enhanced erythrocyte production, a single administration of an adenovirus vector might be suitable. For long-term delivery of Epo, implantation of long-lasting cells engineered to secrete the hormone is more likely, There are still many obstacles to overcome before Epo gene therapy becomes routine in clinical practice. Before it is used on a large scale as a replacement for injections of rHuEpo, it will have to demonstrate major clinical and economic benefits.

8 Naffakh. N. et (,1. ( IwFi; Sustained deliver! gous implants

of genetically-mob,.. ‘:%d

of er~t~tro~oie~~~

in

skin ~brob~asts. PIW.

I’. s. .“L.92. 31943198 9 Hammori. Y.. Samal. B.. Tian, J. ad Kc&.,. L. ( 19%) Tzsistent eryt b! mpoblast transfer oferythropoietin E NA. Hum. 6lw 711e, >. ! W-1

3%

10 Naffakh. N. et al. (1996) Long-term secretion of tbera~eMt~c proteins !rti? genetically-modified skeletal muscles, Htmt. Cow Ther. 7. I 1-2I II

Osborne. W.A. et al.

( 1995) Gene therapy for long-term expression of erythro-

poietin in rats, Proc. Natl. Acad. Sci. U. S. A. 92.8055-8058 12 Descamps. V.

etul.(1995) Organoids direct systemic expressio

in mice. Cole T/lo: 2.41 l-41 7 13 Clowes, M.M. et al. (1994) artery seeded with smoo human genes, J. Clint. best. 14 Hamamori. Y., Samal, B.. Ti er_ythropoietin gene in a mouse 15 Moullier. P. et al. (1995) Long term delivery of lysoso modified fibroblasts in dogs, Nut. Med. I. 353-358 16 Yang. Y. et al. (1994) Cellular

immunity

to viral anttigens limits El-delete

adenoviruses for gene therapy. Pruc. Nutl. Amd. SC;. U. S. A. 9 I.4407331I 17 Tripathy. S.K. et al. ( 1994) Stable delivery of physiologic levels of re~ombi~a~~t erytbropoietin

to the systemic circulation by i~tramuscMtar

injection of repli-

cation-defective adenovirus, Pm. Nat/. Acad. Sri. U. S. A. 9 1, I 155718 Setoguchi. Y., Danel, C. and Crystal, R.J. (1994) §ti~~~~t~o~

How can the time courseof expressionof the transgenefor thropoietin(Epo) be controlledtp obtain either sustainedor transientstimulationof exythropoiesis,dependingon the clinical situation? What is tbe best approachfor regulatingexpression levels

eve levels of sustainedhormone treatmentof hemoglobinopathies? or systems(e.g. adeno-associatedvuus-derivedor non-viralvectors)perform in viva for Epo gene What are the theoretical and actual clinical and economic benefitsof transferof the genefor E&ocorn* withinjections of thepurifiedrecombinanthormone?

erythropoietin gene using an adenovirus vector, Blood 84.2946-7453 19 Tripathy, S.K.. Black, H.B.. Goldwasser. E. and Leiden, J.M. ( 1996) llmmune responses to transgenewencoded proteins limit the stability injection of replication-defective

20 Descamps. V. et al. ( 1994) Erythropoietin

gene transfer and expressive in adult

normal mice: use of an adenovirus vector, Hunt. Gnte Ther. 5,979-985 21 Barr. D. et al. ( 199s) Strain related variations in adenovirally-mediated competent and immunodeficient

inbred strains. Gcnc T/w 2.

22 Scharfmann. R.. Axelrod. J.H. and Vcmla. I.M. of retrovirus-mediated

23 Pugh. B.F. and Tjian, R. (1991) Transcription requires a multisubunit TFIID

J.W. and Ersbach.

J.W. (1990)

The use of recombinant

erythropoietin in humans. in Molecular Cowo; Symposium

148) (Bock. G. and Marsh. J.. eds).

ofHcmopo;esis

human

(Ciba Foundation

pp. 186-200, John Wiley & sons

2 Koury. M.J.and Bondurant, M.C. (1992) The molecular mechanism of erythropoietin action, EM J. Biochem. 210.649-663 3 KouQ’.ST.. Bondwant. M.C. and Koury, M.J. (1988) Localization oferythropoietin synthesizing cells in murine kidney by in s&u hybridization, Blond 7 I, 526527

Letrkentia 6, 107-

T.P.. Clift,

R.E.

( 1992) Fatal polycythemia induced

gene in hematopoietic Cek improves murine @ thalassemia, Blood 84.928-933 7 Skew. L.C.. Burkhart. B.A. and Johnson. EM. (1983) thalassemia. Cell 34. 1043- 1052

A mouse model for p

cells.

and Cottrel, M.B. (1992) Large, chronic doses of in mice, Blood 80.352-358

( 1988) Anemia lessens

II. S. A. 85.6142-6146 27 Madan, A. and Curtin. PT. (1993) A 24-base-pair erythropoietin

sequence 3’ to the human

gene contains a hypoxia-responsive transcriptional

enhancer.

Pm. Natl. Arad. Sri. U. S. A. 00. 3928-3932 28 Wang. G.L.. and Semenza. G.L. ( 1993) General involvment of hypoxia-inducible 90.4304-4308

348

production by hematopoietic

injury and hypertension in rats with reduced renal mass, PwL.. Nat/. A(.ud. Sci.

factor 1 in transcriptional

J.L. et 01.(1994)Retrovirusmmediated transfer of the erythropoietin

promoter

and its prevention with recombinant human erythropoietin worsens glomerular

5 Karhn. S. (1991) lkatment of genetic defects in hematopoietic cell function by gene transfer. Blood 78.2481-2492

111

from a TATA-less

115

4 Erslw A.J. (1991) Erythropoietin. Ne\\* ,%,I$. J. Med. 324. 1339-1344

6 Vikval.

Pm~. Nut/.

complex, Gejfes De\*. 5, 19351945

26 Garcia. D.L.. Anderson. S., Rennke. H. and Brener. B.M.

Adamson.

ISl-l 55 in vivu expression

gene transfer in mouse fibroblast implants.

erythropoietin cause thrombocytopenia

f

( I991) Long-term

Acud. Sci. U. S. A. 88. J626-t630

25 McDonald,

References

trans-

gene expression from mouse hepatocytes irr viva: comparison between immuno-

in mice by dysregulated erythropoietin manuscript and helpful discussions.

ofgeneexpression after

adenovirus vectors, Nut. Med. 2.545-550

24 Villeval. J.L.. Metcalf, D. and Johnson, G.R. Acknowledgements. We thank J.M. Heard and Y. BeuLard for critical reading of the

of ergt

by in uivo gene therapy: physiologic consequences of transfer of the human

response to hypoxia. Pm.. Nutl. Acad. Sci. U. S. A.

29 Madan, A.. Lin. C.. Hatch. S.L.. II and Curtin, PT. (1995) Regulated basal, inducible, and tissue-specific human erythropoietin

gene expression in trans-

genie mice requires multiple cis DNA sequences, Blood 85,2735-274 30 Gossen. M. et al. (1995) Transcriptional malian

cells,Science268. 1766-1769

activation by tetracyclines

1 in mam-