Design of retrovirus vectors for transfer and expression of the human beta-globin gene.

Design of

Retrovirus Vectors for Transfer and Expression of the

Human ,B-Globin Gene

A. DUSTY

MILLER,'*

M. A.

BENDER,"12

EDITH A. S. HARRIS,' MICHAEL KALEKO,' AND RICHARD E. GELINAS'

Department of Molecular Medicine, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle,

Washington

98104,1

and

Department

of Pathology, University

of Washington,

Seattle,

Washington

981952

Received 15April 1988/Accepted 13 July 1988

Regulated expression of the human

0-globin

genehas beendemonstrated in cultured murine erythroleuke-mia cells and in mice after retrovirus-mediatedgenetransfer. However, the low titer of recombinant viruses

described to dateresultsinrelatively inefficientgenetransfer, whichlimitstheirusefulness for animal studies and forpotentialgenetherapy in humans for diseasesinvolvingdefective

0-globin

genes.We foundregionsthat interfered with virus production within intron 2 of the j3-globingeneandonboth sides of thegene.Theflanking regions could be removed, but intron 2was required for ,-globin expression. Inclusion of ,I-globin introns necessitatesanantisenseorientation of the genewithintheretrovirus vector. However, wefound noeffectof the antisense Il-globin transcription on virus production. A region downstream of the

P-globin

gene that

stimulatesexpression of thegeneintransgenic micewasincludedinthe viruseswithout detrimentaleffectson

virus titer. Virus titersofover106 CFU/mlwereobtained with the final vectordesign, which retained the ability todirectregulated expression of human ,-globin in murineerythroleukemia cells. The vector also allowed transfer andexpression of the human ,I-globingeneinhematopoietic cells (CFU-S cells)inmice.

Anunderstanding of the

0-globin

geneand itscontroland the existence of a variety of human diseases caused by

defects in the

P-globin

gene have led to consideration of disease treatment by transfer of a normal gene into bone marrowof affected individuals. Retrovirusvectorscurrently offer the bestvehicle forgenetransferintomarrowcells, and vectorscarrying theP-globingenehave been usedtotransfer the,-globin geneinto murineerythroleukemia(MEL) cells, human erythroid progenitor cells (BFU-E cells), and bone marrow ofmice. MEL cellsare arrestedatarelatively late

stage of erythroid development, but can be induced to differentiate by using a variety of inducers (9, 18). During differentiation, a large increase in mRNA and protein pro-duction from the endogenous globin genes is observed. Following retrovirus-mediated transfer of the human ,B-globingene into MEL cells, mRNAandprotein production from theintroduced human 3-globingene was inducible (4, 7, 13, 15) and intwostudieswasmadeatlevelsapproaching that of the endogenous mouse genes on a per-gene-copy basis (4, 13). Transfer of the

P-globin

gene into human erythroid progenitor cells (BFU-E) resulted in production of RNAfrom the transferred humangene at5% of the levelof theendogenousgene (4). Retrovirusvectors havealso been used totransfer the human P-globin gene into mouse bone marrow. Infusion ofinfectedmarrowintolethally irradiated mice resulted in tissue-specific expression of the gene in erythroid cells, albeit atlevels 100-fold lowerthan those of theendogenous mousegenes(8).

One problem with current vectors which severely limits theirusefulness istheirrelatively low titer, from 7 x 103to 5 x

105

CFU/ml(4,7, 8, 13, 15). The low frequency ofgene transfer into bone marrow cells capable of reconstituting

mice(18 DNA-positive animals of 104 tested) (8) isapossible consequence of this problem. Amphotropic retroviral

vec-tors have been used to transfergenes into human

hemato-poietic progenitorcells,including erythroidprogenitors,but

* Corresponding author.

only the highest-titer vectors (106 to 107 CFU/ml) yield useful frequencies of transfer (11, 12). Indeed,wefindvery poorinfection of human BFU-E cells by usingour

P-globin

vector(4). Thus, development ofhigher-titerviruses

carry-ing the ,B-globin gene is requiredto facilitate gene transfer experiments and forpotential application of these techniques togenetherapy in humans.

Inthisstudy, we examined thefactorsleadingtothelow titer of ,-globin viruses and made alterations in the virus which resulted in a substantial increase in titer without

affecting'-globingeneexpression. In addition,we incorpo-rated into the viruses a region downstream of the human

P-globin

genethatisimportant for regulated human

P-globin

expression in transgenic mice (3, 14, 26). The new virus

allowed efficient infection of murine hematopoietic cells (CFU-S cells) and human 1-globin expressionwas detected

in resultant hematopoietic colonies in mice.

MATERIALS AND METHODS

Cellculture.CellsweregrowninDulbecco modifiedEagle medium with high glucose (4.5 g/liter) supplemented with

10%calfserum

(*2

cells)or10% fetalbovineserum(all other

cell lines). Concentrations of G418 are calculated by using

the weight of dry powder, of which about 50%wasactive. Previously described cell lines include PA317 (19) (ATCC CRL 9078), adenine phosphoribosyltransferase-negative

(APRT-) tetraploid MEL cells selected tobe semiadherent (gift ofP. Mellon, originally obtained from A. Deisseroth), thymidinekinase-negative (TK-) NIH 3T3 (20), and 42 (17). PA317retroviruspackaging cells used herewereeither from an early passage of the cell line or were reselected in

hypoxanthine-aminopterin-thymidine medium as described

previously (5). For induction ofdifferentiation, MEL cells

were seeded at 5 x 104 cells per ml in medium containing

3 mM N,N'-hexamethylene-bisacetamide (HMBA; Sigma ChemicalCo., St. Louis, Mo.)on day1. Onday 4, medium wasremoved from the cellsfollowing low-speed

centrifuga-tion andthe cellswere resuspended in the samevolume of

4337 0022-538X/88/114337-09$02.00/0

Copyright C1988, American Society for Microbiology

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(2163) (2422) (2482) (3287) Pstl Pstl AvrIl Xbal

I

i

l

II

I 11

I

_

HP-VIRUSES

F-SX-VIRUS SA-VIRUSES

FIG. 1. Human P-globin gene and surrounding sequences.

Se-quencenumbersaregiven relativetothe RNAcapsiteat +1.

13oxes

indicate exons, and hatched regions indicate the protein-coding

region.ABglII site (AGATCT)wasinserted between bases+40and

+41,asindicated,tomarkthegene(4). Landmark restriction sites aregiven, andconsensus poly(A)signals (AATAAA) and orienta-tion of the signalsareindicatedbyarrows.Insertsusedin different vectorsarediagrammed below the--globingene.

Retrovirus vectors. Retrovirus vectors expressing the neomycin phosphotransferase (neo) drug resistance gene wereusedto transferP-globingenes. The prefix pindicates

the plasmid form of the virus. Retrovirussequencenumbers are as described previously (27). The vector pLNL-XI:C contains theneogeneinserted intoaDNA clone ofMoloney

murine leukemiavirusinplace of Moloney murine leukemia

virus sequences from 1039 to 7673 and contains XhoI, HindIII, ClaI restriction sites between theneogeneand the 3' long terminal repeat (LTR) for insertion of additional genes(5). ThevectorpLNSL7 contains (in thedirection of transcription) the 5' LTR through base 544 from Moloney murine sarcoma virus, bases 569 to 1038 from Moloney murineleukemiavirus,toBglII-to-SaII fragment from

trans-poson Tn5 containing the neogene (2), aPvuII-to-HindIII fragment from simianvirus40 containingthesimianvirus 40

early promoter, unique StuI, AvrII, HindIII, and ClaI sites for inserting cDNAs, and bases 7764 through the 3' LTR from Moloney murine leukemia virus. In addition, thegag

startcodon in pLNSL7 waschanged from ATGtoTAG to prevent possible gag protein translation (5). Both

LNL-XHC and LNSL7 were produced at equal titer by using retrovirus packaging cell lines.

3-Globingenes modifiedby insertion ofaBglII site as a

transcriptional marker (Fig. 1) (4)wereinserted into pLNL-XHC between the neo gene and the 3' LTR (pLNB*HP, pLNB*HP

MG',

and pLNB*HP MG-) and into pLNSL7 between theneogeneandthe 3' LTR inplaceof the simian virus 40 promoter (all other ,-globin vectors). Retrovirus vector names indicate the order ofgenes in the virus and important features of the vector. For example, LNB*HP indicates a retrovirus vector consisting ofa viral LTR (L) driving neo (N) followed by a transcriptionally marked ,-globin gene (B*) contained in an HpaI-to-PstI (HP) ge-nomic fragment. The vectors LNB*MG+, LNB*MG-, and LNB*WT-describedinapreviouspublication (4)have been renamed LNB*HP

MG',

LNB*HP MG-, and LNB*HP, respectively, to allow consistent nomenclature use in this

report. To make LNB*SA RP (reversed promoter), the SnaBI (-265)-to-BgIII (+43) fragment containing the ,3-globin promoter(Fig. 1) in LNB*SAwas excised and

rein-serted inreverseorientationby usingaBgIII linkertoadapt the blunt-endSnaBI sitestotheBglIIsites. TheBglIIsite is notpresentinthenormal 3-globingenebut ispresentinthe marked gene (Fig. 1). To make LNB*SA P- (promoter minus), the same fragment was removed from LNB*SA entirely, again using a BglII linker to join the SnaBI and BglII sites.

Generationof virus from retrovirus vector constructs. Virus

was generated from plasmid constructs as previously de-scribed(22). Briefly, plasmids containingthe viral constructs

were transfected into p2 ecotropic retrovirus packaging cells,and after 2daysviruswasharvested and used to infect PA317 amphotropic retrovirus packaging cells. The cells

werethen seeded into selectivemedium,andclonal celllines containing single integrated proviruses were isolated. The structures ofintegrated P-globin viruses were analyzed by Southern analysis (16) with a ,-globin minigene probe. By

usingrestriction enzymes that cleave only in each LTR of the virus(KpnI)andotherenzymesthat cutoncewithin and at sites presentat both ends of the 3-globininsert (BamHI andHindlll),weconfirmed that thefragmentsizesproduced by the integrated provirus matched those of the original plasmid construct.

Virus assay. Virus was harvested from virus-producing cells by incubating confluent dishes of the cells with fresh

medium for 16 h and thenremoving the medium and sub-jectingit tocentrifugationat3,000 x gfor 5minto remove

cells and debris. Forassayof virusescarryingtheneo gene, recipientcellswereseeded at5x 105per60-mm dishonday

1.Onday 2,themediumwaschangedto mediumcontaining 4 ,ug ofPolybrene perml andvarious amountsoftest virus

wereadded. Onday 3, the cellsweresplit1:10 into medium containing1mgofG418 (about50%active)perml. Colonies

were stained and counted on day 9. Amphotropic helper virus was measured by the S+L- assay as previously

described (20). The presence of helper virus in mice was

monitored by using the XC assay (23) as previously de-scribed(20), exceptthatrecipient NIH3T3 TK- cellswere

cocultivated with 10

[lI

of fresh blood instead of virus-containing medium for 16 h and blood cells were washed fromtheplate beforethe cells were trypsinizedfor the XC

assay.

Infection of murine bone marrow cells and CFU-S assay. 5-Fluorouracil (150 mg/kg; LyphoM'ed Inc.) was adminis-tered intravenously to 6- to 8-week-old female C57BL/6J mice(Jackson Laboratory, BarHarbor, Maine). Micewere

sacrificed48'hlaterbycervicaldislocation,andmarrowwas

flushed from femurs and tibias by using cocultivation

me-dium (Iscoves medium with 10% heat-inactivated fetal

bo-vine serum [Hyclone Laboratories, Logan, Utah], 10% WEHI-3cell conditionedmedium,4,ugofPolybreneperml, penicillin, and streptomycin). Marro'w wascocultivated on

subconfluent monolayers of irradiated (2,100 rads) virus producercells for24 h.Hematopoieticcellswerewashed off

themonolayerand cultured for 36 to 48h in the presence of

1 mgofG418 per ml. Marrow was washed with and

resus-pended in Hanks buffered saline solution withoutcalcium, magnesium,orphenolred andinjectedintofemaleWBB6F1/ J-W/Wv mice (Jackson Laboratory) which had received

either no irradiation or400 rads. At 12 days postinjection, individual CFU-S colonies were dissected out and

dissoci-ated and total nucleic acid was isolated(4).

RESULTS

LNB*HP viruses: j8-globinviruses containing the HpaI-to-PstIgenomic fragment. Figure1 depictsthe human ,-globin gene and surrounding sequences. Before insertion into

vi-ruses, the ,-globin gene was modified by insertion of a

6-base-pair BglIIsite into the nontranslatedregionofexon1 (Fig. 1). This wasdone to allow measurement of transcrip-tion from the gene in human cells already expressing the endogenous ,-globin gene. When we began these

experi-(-814)(-615) (-265) (+1) (479)

HpalSphI SnaBi CAP BamHI

A

K_

m~IIER

r.

Bglll - .

INSERT

(1396) EcoRI

7

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TABLE 1. Characteristicsof PA317 cells containing ,-globinvectorsa

Titer(CFU/ml)in: P-GlobinRNA

Vector Correct expression in

structure? PA317

*2

PA317 cells?

Parental

neo-Virus >90% 1 x 107 1 X 107

LNB*HP 3/20 5 X 104 5 X 105 +

LNB*HPMG- 6/8 2 x 106 NDbb

LNB*HPMG+ 7/8 4 x 106 ND

-LNB*SX 2/13 2 x 104 4 x 105 +

LNB*SA 13/15 2 x 105 4 x 106 +

LNB*SA 1-2+ 7/8 5 x 105 ND +

LNB*SA 1+2- 7/8 2 x 106 ND

+/-LNB*SA MG 4/4 4 x 106 ND

-LNB*SA RP 7/10 3 x 105 ND

-a Clon-alPA317 celllinescontaining the indicated retrovirusvectors were

generatedasdescribed in Materials and Methods. The number of cloneswith

the correctproviral struicture is indicated as the ratio of clones with the correct structure tothe totalanalyzed. The highest titer obtained fromPA317 cellscontaininganunrearrangedvirus is indicated for eachvector.Synthesis ofP-globin RNA in PA317 cells containing each vector is indicated, as

determined byNorthern analysis withaP-globinprobe. Virus from PA317

cells was used toinfect s2cells, infected cloneswereisolated, andthe best

titer obtained from%P2cellscontaininganunrearrangedprovirus is indicated.

Atthetime ofassay,the

q$2

cellswerehelper virus free (<10PFU/ml), as

measured by theXCassay (20,23).After continuedpassage,helper viruswas

detected,ashaspreviously been reported fortheparental neo-virus (22).

bND,Notdetermined.

ments, the region from HpaI (base -814) to PstI (base

+2163) appeared to be sufficient for developmentally regu-lated tissue-specific expression of the gene in transgenic mice, andweinserted this fragment intoa selectable retro-virus in reverse orientationwith respect toviral transcrip-tion. We also inserted the same fragment containing an

intronless

3-globin

gene, or minigene, into the selectable retrovirus in both orientations. Virus generated by using

PA317 retroviruspackagingcells(19) was madeathightiter from vectors carrying the 3-globin minigene in either the

forward (LNB*HP

MG')

or reverse

(LNB*HP

MG-) orien-tation, while the vectorcontaining the ,B-globin gene in the reverse orientation (LNB*HP) yielded virus with a much lower titer (Table 1) (4). Conversely, transcription of

P-globin was only detected in fibroblasts, MEL cells, or

BFU-E colonies infected with the vector

carrying

the

P-globin gene with introns and not in cellsinfected with either

vectorcarrying a ,-globin minigene (4). Thus, transfer ofa transcriptionally active,B-globingeneappearedtobe

incom-patible with producing high-titervirus.

To understand how thedifferent,-globin inserts affected theproduction of virus,weexamined RNAfromPA317cells containing the different vectors and fromvirions produced by these cells (Fig. 2A).

PA317

cells infected with the

parental neo-virus contained two predominant viral RNAs

(depicted in Fig. 2B), a full-length viral RNA which is

packaged into virions and a spliced RNA which is not

packagedbecause it lacks the signal requiredforpackaging

(1, 5). In contrast, PA317 cells infected with the ,B-globin viruses contained multiple viral RNAs, and virions produced

by the cells contained atleast two RNAs, of which only a minorportionwasthefull-length viral RNA(Fig. 2A). The

effectsontiter thatweobserved(Table 1) correlated with the

proportion of full-length viralRNAobserved in cells and in virions, which was reduced for the minigene viruses and was

barelydetectablefor the LNB*HP virus(Fig. 2A). Thus, the

low titer ofthese

P-globin

viruses was apparently due to the

low abundance of

full-length

viral RNA in

virus-producing

cells and in virions

produced by

the cells.

To understand the reasons for the low abundance of full-lengthviralRNA,we

analyzed

the

transcription

patterns of the vectors, and we propose the

transcription

patterns

shownin

Fig.

2B to

explain

the RNA data in

Fig.

2A. The

transcripts

found in PA317 cells

harboring

the LNB*HP

MG'

virus can be correlated with known

transcriptional

signals

in the virus. There are two

poly(A)

signals

in this

virus, onein the 3' LTRandone at the endofthe

3-globin

minigene

where

polyadenylation

of

3-globin transcripts

nor-mally occurs

[signals

labeled

(A)n,

Fig. 2B].

In

addition,

splicing

can occurbetween the 5' LTR andneosequencesin the

parental

neo-virus

(1,

5).

Thus,

we expect four viral

transcripts

in cells

containing

LNB*HP

MG'

(Fig. 2B).

Indeed,

threebandsweredetected in PA317 cells

containing

the LNB*HP

MG'

virus

(indicated

by

dots in

Fig. 2A)

corresponding

inorderof

decreasing

sizeto

expected

RNAs a,b

plus

c, and d

(Fig.

2B),

assuming

that theabundant28S rRNA

(4.5

kilobases

[kb])

caused bands b and c to

comi-grate.

Analysis

ofcellularRNAwithneoor

,-globin probes

revealed a similar

pattern

of bands. The sizes of the two RNA speciesinvirions wereconsistent with

expected

pack-aging

of

unspliced

RNAsaandc

(Fig. 2B).

Thus,

viral RNA

species

found in cells

containing

LNB*HP

MG'

and in virions

produced

by

the cells match the

predicted

RNA

species

depicted

in

Fig.

2B. In

addition,

since RNA

corre-sponding

to the

full-length

viral

transcript

was observed in

cells and in

virions,

this indicates that

transcription

can

occuracrossthe

P-globin

poly(A)

signal

presentin the virus. In the LNB*HP MG-

virus,

the

,B-globin minigene

is inserted inreverseorientationwith respecttoviral

transcrip-tion.Noknown

poly(A)

or

splicing signals

arepresentin the inserted

P-globin

sequences, but there is one consensus

poly(A)

signal (AATAAA)

in the

3-globin

geneandfoursuch

signals

clustered at the 3' end ofthe insert

(asterisks,

Fig.

2B). 1-Globin

andneo

probes

revealed

transcripts

of similar

size in LNB*HP MG--infected cells

(indicated

by

dots in

Fig. 2A)

which

correspond

in order of

decreasing

size to

proposed

transcripts

a, c,

b,

and d

(Fig. 2B), although

as noted above the

separation

ofbands b and cis

complicated

by

their

proximity

tothe 4.5-kb rRNA band. Two

transcripts

weredetected invirions harvested fromPA317 cells

contain-ing

LNB*HP MG-

(Fig.

2A),

and their sizes matched

proposed

transcripts

aand c

(Fig. 2B). Thus,

RNA

species

found in PA317 cells containing LNB*HP MG- and in

virions

produced by

the cells can be accounted for

by

splicing

known to occur in the

parental

neo-virus

and the presence of an additional RNA termination

signal

in the

P-globin insert,

which coincides with several consensus

poly(A)

signals.

Note that

analysis

of RNA in LNB*HP

MG'

and LNB*HP MG- virions reveals

equivalent-size

bands which

correspond

to the

predicted full-length

viral

transcript,

as

expected

since the virusesare the same

size,

anddifferent-sizesmaller bands

corresponding

to

early

RNA

terminationat different

positions

in thetwoviruses.

Finally,

one

might

expectthat the patternof RNA

species

observed fortheLNB*HP virus would be similartothatof

LNB*HP

MG-,

but shifted up 1 kb in size to reflect the additionof the

3-globin

introns. The observed pattern was

more

complex

(Fig. 2A),

but consistent with premature RNA termination within intron 2 of the

3-globin

gene

(Fig. 2B).

Proposed

RNAs e and f

(Fig. 2B)

predominated

in PA317 cells

containing

the virus

(lower

two

dots,

Fig. 2A),

and RNA

species

e wasthe

major

RNAinvirions

produced

from these cells

(lower dot, Fig. 2A). Thus,

it appears that

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(50O (50(

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CL 0-Q n- a- L1

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NEO-VIRUS

a

b

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=~~~~~~~~~~~~~~~~~~

LNB*HP MG+

a

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LNB*HP

MG-a

b

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FIG. 2. Analysis of RNA from,-globin vectorscontaining the Hpal-to-PstI ,-globin fragment. (A) RNAsfromPA317cells containing

P-globinvectorsandfrom virionsproduced by the cellsweresubjectedtoelectrophoresis inagarosegelscontaining formaldehyde and probed

by usinganHpaI-to-PstIp-globinminigene probeor aneoprobe bystandard methods (16). The positions of the human,-globinmRNA(hP*), thefull-length RNAs from the minigene viruses (MG), and the full-lengthRNAfrom theintron-containing,B-globinvirus (HP)areindicated.

Positionsofthe28S (4.5 kb) and 18S (1.8 kb)rRNAsareindicated,andspecific bands discussed in thetextareindicated by dotstotheleft

of eachlane.(B) Proposed RNA speciestranscribed from retrovirusvectors.Approximate sizes of theRNAsaregiven in kilobases assuming

a200-base-pair poly(A) tail.Asterisks indicateconsensuspoly(A) signals (AATAAA)inthetranscriptional orientation of the viralpromoter,

(A)nindicatesknownpoly(A) sites in the orientation of the viralpromoter,andarrowsindicatepromoters.

LNB*HP GENOMIC RNA

(HP)

LNB*HP MG GENOMIC RNA (MG)

-

hp*

d

(A)

r-ol NEO 11 ri

LTR,.

d.No\"NINIIINI. ri A

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MG-4.5

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z z

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r-t---4.5kb

-

1.8

kb

-PROBE

FIG. 3. Comparison of RNA in PA317 cellscontainingLNB*HP

orLNB*SX.Northern (RNA)analysis wasperformedasdescribed

in the legend to Fig. 2A. One PA317 clonal cell line containing LNB*HP and three independent clones containing LNB*SX were

analyzed. Thesizesof the ribosomal markers and thepositionof the

human 3-globin(h,*)RNAareindicated.

sequencesin intron2in thereverseorientation

P-globin

gene

act toreduce the amountof full-length viral RNAin PA317 cellscontaining theLNB*HP virus and in virions produced by these cells. RNAwith the size expectedfrom the

P-globin

gene was detected in cells containing this virus. This

P-globin transcript was not detected in virions produced by these cells, even though it is transcribed in an antisense

orientation with respect tothe viralgenome and thus could hybridize to viral RNA. In summary, the low titer of the

P-globin

viruses appears to be due to early termination of viral transcription, which reduces the amount offull-length viral RNAin virions.

LNB*SX: a

0-globin

virus containing the SphI-to-XbaI

genomic fragment. Inanattempt toimprove the titer ofvirus containingthe,-globingene, weconstructedavirus thatdid

not contain the HpaI (-814)-to-SphI (-615) region of the

gene which contained the four consensus poly(A) signals

(Fig. 1), in which early termination of RNA transcription appeared to occur. In addition, evidence had accumulated

suggesting involvement of sequences downstream of the

gene in ,B-globin gene regulation (3, 14, 26). Thus, we

included these sequences in the LNB*SX virus, which

contains the region from SphI to XbaI inserted in reverse

orientation intoaneo-virus. PA317celllines containingthis

vector were generated and screened as for the LNB*HP

virus. Unfortunately,weobservedratesofrearrangementof theLNB*SX virus inPA317cellsthatwere ashigh asthose

seen with LNB*HP. In both cases, the rearranged viruses

were smallerthan theparental virus andhad suffered

dele-tions of 3-globinsequences.ThebesttiterfromaPA317cell

clonecontaininganunrearrangedLNB*SX proviruswas2 x

104, evenlowerthatobtained with LNB*HP(Table 1).

Analysis of RNA in PA317 cells containing either the LNB*SXorLNB*HP virus(Fig.3)providedanexplanation

for the low titer of theLNB*SXvirus. Transcriptsfromthe

viral LTR through the ,B-globin gene were reduced in

LNB*SX-infected cells in comparison with those in

LNB*HP-infected cells. In contrast, the amount of RNA

transcribed from the

,-globin

gene was similar in cells infected with either virus. Since the

probe

used in these

experimentswas a

3-globin

minigene

probe

(positions

-814 to +2163, Fig. 1), it is

likely

that an additional RNA termination signal in the PstI-to-XbaI

fragment

present in

LNB*SX, possiblythe

poly(A)

signal

at

position

+2637

(Fig.

1), leadsto terminationbefore the

region

recognized

by

the probe. Apparently, the titer ofthe LNB*SX virus wasnot improvedcomparedwith that ofLNB*HP

because,

whilewe may have removed one termination

signal,

we introduced

another.

LNB*SA: a

0-globin

virus

containing

the

SphI-to-AvrII

genomic fragment.

Assuming

that the

poly(A)

signal

at

position +2637

(Fig. 1)

was

responsible

for the low titer of the LNB*SX

virus,

we deleted this

signal

in our next

,B-globin

virus,

LNB*SA,

which contains the

SphI

(-615)-to-AvrII(+2482)

region

containing

the

P-globin

gene

(Fig.

1)

in reverse orientation. This virus still contains the

region

downstream ofthe

3-globin

gene involved in

regulation

of the gene, whichhasbeenlocalizedbetweenbases+2163 and +2422(3). Incontrasttoresults obtainedwithLNB*HPand

LNB*SX, most of the PA317 cell lines infected with

LNB*SA virus contained

unrearranged

proviruses,

and the

highest titer observed was 10-fold

higher

than that of LNB*SX and4-fold

higher

thanthat of LNB*HP

(Table

1).

Therefore, we conclude thatsequences inthe

regions

flank-ingthe

P-globin

genefrom

positions

-814to -615 andfrom

positions +2482 to +3287 inhibit virus

production

when inserted into aretrovirusinreverseorientation. RNA

anal-ysissuggeststhatthis resultisduetoprematuretermination

of RNA

transcription,

which reduces the amount of

full-length viral RNA and thus also reduces the titer of virus produced.

Role of

I-globin

introns in LNB*SA virus. The titer

ob-tained from theLNB*SAvirus

(2

x

105

CFU/ml)

wasbetter than that of other viruses

containing

a

P-globin

gene with introns but was still far from the titer obtained with the

minigeneviruses(2 x

106

to4 x 106

CFU/ml).

Todetermine which intron was

responsible

for the decreased

titer,

we constructed three new viruses with structures identical to thatof LNB*SA exceptthat LNB*SA 1-2+ lacked intron

1,

LNB*SA

1+2-

lacked intron

2,

and LNB*SA MG lacked

both

P-globin

introns. Most of the PA317 lines

generated

fromthesevirusescontained

unrearranged

proviruses,

as we found for the

parental

LNB*SA virus

(Table 1).

The mini-gene virus LNB*SA MG

(which

does not contain

P-globin

introns)hadthe

highest

titer,

20-fold

higher

than

LNB*SA,

a result similartothat obtained

previously

with the LNB*HP

viruses (Table 1). Virus titersfrom the LNB*SA 1-2+ and

LNB*SA 1+2- viruses were intermediate to those of LNB*SA and LNB*SA

MG,

but

clearly

the presence of

intron 2 best correlated with reducedvirus titer

(Table

1).

Wenext

analyzed

the RNA in the PA317 cells

containing

LNB*SAanditsderivatives

(Fig.

4).

A

transcript

of the size

expected

from the

0-globin

gene was present in

LNB*SA-andLNB*SA 1-2+-infected cells butnot inLNB*SA 1+2--or LNB*SA MG-infected PA317 cells.

Quantitation

of

P-globin

transcripts

by

an RNA

protection

assay revealed similar amounts in LNB*SA- and LNB*SA

1-2+-infected

cells, 7% of this level in LNB*SA 1+2--infected

cells,

and

only 1%in LNB*SA MG-infected cells.

Thus,

transcription

of the

3-globin

gene

strongly

depended

on the presence of

P-globin

intron 2.

Expected

full-length

proviral

transcripts

(indicatedbydots in

Fig. 4)

wereabundant in LNB*SA MG-and LNB*SA 1+2--infected

cells,

were

greatly

reduced in

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[image:5.612.128.239.74.287.2]

1-2- 1+2- 1-2+ 1+2+

-4.5kb

-1.8kb

-hp

A

-PROBE

FIG. 4. Northern analysis of RNA in PA317 cells containing LNB*SAanditsderivatives lacking one or more introns. RNA from independentPA317 clonal cell linescontainingLNB*SAMG (1-2-), LNB*SA 1+2- (1+2-), LNB*SA 1-2+ (1-2+),andLNB*SA(1+2+) wasanalyzed as described in the legend to Fig. 2A. The sizes of rRNAs and the position of the human

P-globin

mRNA

(hp*)

are shown, andthefull-length viral RNA species for each cell line is indicated byadottothe left ofeachlane.

LNB*SA1-2+-infected cells, and were barely detectable in

LNB*SA-infected cells. As anticipated, the amount of

full-length viral transcript present (Fig. 4) correlated with the titer ofthe virus(Table 1). Finally, the amount of full-length

viral transcript, and thus the titer of virus, was inversely

related to the amount of ,-globin message present in the

virus-infected cells.

Effect of

,3-globin

gene transcription on virus production. Theresults presented above lead to theunfortunate

conclu-sion thatthe titerof virusescontaining the

3-globin

gene in reverse orientation is inversely related to the ability ofthe virus to make a

3-globin

mRNA. We considered three

possible explanations for this effect. (i) The introns

them-selves, in particular intron 2, contain signals that inhibit

production of thefull-length viral RNA; (ii) the presence of anactivepromoterinterfered withviraltranscription; or (iii) thepresence of the,B-globin mRNA, which has an antisense

orientation with respect to viral mRNA, caused rapid

deg-radation oftheviral RNA or in some other manner affected

accumulationof viral RNA. To test the latter two

possibili-ties,wealtered LNB*SAtopreventproductionof antisense RNA. In LNB*SARPthe ,-globin promoter was reversed, and in LNB*SA P- the promoter was removed entirely.

PA317 celllines containing LNB*SA RP were made and screened asdescribed above. Both the percentage of clones

containing an unrearranged virus and the titer of virus produced from the best clone were similar to results ob-tained with the parental LNB*SA virus (Table 1). As

ex-pected, RNA ofthe size expected from the ,B-globin gene was not detected in PA317 cells containing LNB*SA RP by using a ,-globin probe (data not shown). Viral transcripts detected were similar in sizes and abundance to those of LNB*SA (data not shown). Thus, the low titer of the LNB*SA virus was not due to reverse orientation

transcrip-tion(withrespect to viraltranscription) of the

3-globin

gene orto the presence of ,(-globin RNA in the virus-producing cellswhich has an antisense orientation with respect to viral

transcripts.

Next, we tested the ability of pLNB*SA P- to make high-titer virus. For this assay, we used a transient virus production assay in which retrovirus-packaging cells are transfected with the virus constructs, virus is harvested 2 days later, and the titer of the virus is measured. This assay is much faster than the assayused for the virusesdescribed

sofar, and we found agoodcorrelation between the results of the two assays with ,B-globin viruses. The results show that pLNB*SA, pLNB*SA RP, and pLNB*SA P- all pro-duce virusat atiter that is atleast 20-fold lower than that of pLNB*SA MG or the parental neo-virus (Table 2). That the titer of LNB*SA P- was not improved indicates that the presence of an active ,-globin promoter in LNB*SA is not responsible for the low titer of this virus. Therefore, we conclude that the inverse correlation between ,-globin

expression and virus titer observed for the LNB*SA, LNB*SA

1+2-,

LNB*SA 1-2+, and LNB*SA MG viruses does not reflect inhibition of virus production as a result of

P-globin

gene transcription. Instead, we conclude that ,-globin intron 2 contains signals that inhibit transcription of the full-length viral RNA, which results in reduced virus titer.

I-Globin

virus

production

from

*2

retrovirus-packaging

cells. We tested the ability of another retrovirus-packaging cellline,

qi2

(17), to produce several of the vectors. Interest-ingly, the titers of ,B-globin viruses LNB*HP, LNB*SX, and LNB*SA were 10- to20-foldhigher from 4i2 cells than from PA317 cells, even though the highest titer of the parental neo-virus was similar in both cell lines(Table 1). The titer of LNB*SA was 10-fold higher than that of LNB*HP or

LNB*SX, consistent with results found with PA317 cells. Helper virus was not detected in any of these clonal lines at thetimeofassay and thuscannotexplainthehigh titerof the

,B-globinvectors.

Expression of j8-globin gene in infected MEL cells. We

infected MELcells with severalof the ,-globin virusesand measured expression of the transferred

P-globin

gene in

uninducedandinducedMEL cellsby usingan RNA protec-tion assay(Table3). We found that all viruses containing the

P-globin

gene with both introns as well asthe virus

contain-ing only intron 2 (LNB*SA 1-2+) directed similar amounts of correctly initiated ,-globin RNA synthesis in induced cells. In contrast, ,-globin RNA was reduced more than 10-fold in MEL cellsinfectedwith theviruscontainingintron 1but not intron 2(LNB*SA 1+2-) and was not detected in MEL cells infected with the minigene virus. These results

parallelthose obtained with fibroblasts(Fig. 4)and showthat

intron 2 is required for efficient

P-globin

expression. In

addition, ,-globin RNA was induced to similar extents in MEL cells containing all the viruses except for LNB*SA

MG,inwhich

P-globin

RNAwas not detected. Thus,either

[image:6.612.76.272.72.252.2]

,-globinintroncanbe deletedwithout

affecting

the

ability

of

TABLE 2. Effect ofP-globinpromoter alterations on virusproduction'

Vector Transient titer

(CFU/ml) pLNB*SA... 1 x104 pLNB*SARP... 6 x 103 pLNB*SAP ... 1 x 104

pLNB*SAMG... 2 x105

neo-Virus... 3 x 105

"Eachofthe indicatedvectors(10,ug)wastransfected intoP2cellsplated the day before at 5 x 105 per 6-cm dish by using calcium phosphate coprecipitationasdescribedpreviously(21).The cellswerefed 1dayafter

transfection,and after 2daysviruswasharvested andquantitated.

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TABLE 3. P-Globinexpressionin MEL cells' 1-GlobinRNA Avginduced

Vector [% of totalpoly(A)+RNA] RNA level Avg fold

comparedwith induction

Uninduced Induced LNB*SA(%)

LNB*HP 0.0073 0.066 110 9

LNB*SX 0.0031 0.039 70 13

LNB*SA 0.0044 0.058 100 13

LNB*SA1-2+ 0.0017 0.027 50 15

LNB*SA1+2- 0.0003 0.0023 4 8

LNB*SA MG <0.0002 <0.0002 <0.3

p-GlobinRNAproductionwasmeasured ininduced and uninducedMEL

cellsby anRNAprotection assay aspreviously described(4). Results are

averages of twoexperiments for each vector. Cells analyzedarepools of

G418-resistant MEL cells resulting from infection with virus fromPA317cells

containing the indicatedvectors.

the gene torespond toinduction. Finally, these results show that the presence of theadditionalregion downstream of the

P-globin

gene implicated in control of gene expression

(which is present in LNB*SX and LNB*SA but not in

LNB*HP)had nosignificant effect on

P-globin

expressionin MEL cells.

The presence of human ,-globin polypeptide in infected

MEL cells was determined byimmunofluorescence with an

antibody for human

f-globin

which does not recognize

mouse

P-globin

(24). MELcells infected with the LNB*HP

andLNB*SAvirusesdisplayedsimilarfluorescence,with 20 to 40%of the cells in these nonclonal populations showing bright fluorescence (data not shown). Thus, cells infected

with the high-titer LNB*SA virus make human 3-globin polypeptide in amounts similar to those demonstrated

pre-viously fortheLNB*HPvirus (4).

Expression of human I-globin in infected mouse hemato-poietic cells. We used the high-titer virus from

jp2

cells

producing the LNB*SA virus to infect bone marrow from

mice. Infectedmarrow wasinfused intoW/WVanemic mice, and after 12days spleen colonieswereisolated and analyzed for the presence of vector DNA and for human

P-globin

RNA expression. Twelve-day CFU-S colonies arise from

primitive hematopoietic cells which give rise to colonies

containing various hematopoietic lineages, including

ery-throid cells. In separate experiments, 6 of9 and 10 of10 CFU-Scolonies containedaprovirus of theexpected struc-ture (data not shown). Every infected CFU-S colony

con-tained correctly initiated human ,-globin mRNA, although

the level ofexpression varied over100-fold(Fig. 5).CFU-S colonies containavariable mixtureoferythroidand nonery-throid cells, which may account for part of thevariation in

expression. However, we have no evidence that human

3-globin

expression is indeed erythroid specific in these

experiments. No helper virus was detected in similarly

treated miceanalyzed after1 month, showing that infection was notthe resultof virus spread. Thus, LNB*SA was able to infect primitive hematopoietic cells and the transduced

globingene wasexpressed in theresulting colonies.

DISCUSSION

We made a

variety

ofalterations to increase the titer of viruses containing the human ,B-globin gene. Several signals in both intron 2 and regions flanking the

3-globin

gene interfered with the retrovirus life cycle. We could eliminate theinhibitory flanking signals but not the signal(s) in intron 2, because the presence of this intron was required for

:~~~

rt,

I

ME

5.-hf3

CFU-S

FIG. 5. HumanP-globinRNA(h1*)synthesis in infectedCFU-S colonies. Bone marrow was cocultivated with

qj2

cells producing LNB*SA, selectedin G418, and infused into anemic W/WV mice. Twelve-day CFU-S colonies were isolated, and total nucleic acid fromprovirus-containing CFU-S colonieswasanalyzedbyanRNA protection assay (4). RNAfrom aMEL cell line infected with a ,B-globin gene-containingviruswasusedas amarker.

efficient

P-globin

expression. For the virus containing a forward orientation,B-globin minigene (LNB*HP

MG'),

the

interfering signalwas apparently the normal poly(A) signal

present at the end of the

P-globin

gene, which led to decreased full-length virus transcription. Less than full-lengthtranscriptionin other 3-globinviruses alsocorrelated withthe presence of consensuspoly(A) signals (AATAAA)

within thevirus,butcouldbeduetoothersignals for3' RNA

processing orfor termination oftranscription. In addition,

notall consensus poly(A) signals are functional and notall functional poly(A) signals share the AATAAA consensus sequence (6); thus, the elimination of AATAAA signals mightnotbesufficienttoincreasefull-length viral

transcrip-tion.

Weexploredthe effecton

P-globin

expression ofaregion

downstream of the,-globingenethat has beenimplicatedin

3-globin

regulation, but found no effect on expression in MELcellsorin fibroblasts. Inaddition,limitedexperiments

suggestthat thisregionhasnoeffecton,-globin expression

inanimals reconstituted withinfectedmarrow(8). However,

inclusion of this region in the LNB*SA virus does not

appreciably affect virus titer; therefore, we retained the sequence. We hope that regions flanking the globin locus which are responsible for position-independent high-level

expression of

3-globin

in mice (10)can eventuallybe

incor-poratedinto these vectors. At present, however, the local-ization of these elements is not precise enough to allow insertion of the region without exceeding retrovirus size limits.

Problems that have been encountered whenusing

retrovi-ral vectors to transfer genes can be grouped into three

categories: inappropriatesplicing, early termination of viral

L

I

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[image:7.612.318.567.71.325.2] [image:7.612.66.300.84.184.2]

RNA, and competition of multiplepromoterscarried by the viruses (forareview, see reference 25). Since the

P-globin

intronswerefound tobe necessaryfor

P-globin

expression, we were limitedto insertion of thegene in reverse

orienta-tion toavoid removal oftheintrons bysplicing during virus replication. Less than full-length viral transcription was encountered with reverse orientation inserts, which was reduced but not eliminated inredesigned viruses. We have direct evidence that possible competition between viral and ,-globinpromotersorpossibleeffectsofantisenseRNA did not affect virus titer, since reversal or removal of the ,-globin promoter had no affect on virus titer. The diffi-culties encountered with retrovirus-mediated transfer and expression of 3-globin contrast with the ease with which another transcriptionally regulated gene, a rat growth hor-mone minigene, was transferred and expressed after inser-tion into a retrovirus vector(21). Despite these difficulties, systematic modification of viruses containing the

P-globin

geneledtoa 3-globinviruswhichcanbemadeatarelatively high titer. Still higher titers might be achieved if the ele-ment(s)inintron 2 whichinterferes with transcriptioncanbe altered without affecting ,B-globin expression, for example, by site-specific mutagenesis of the presumed poly(A)signal. In generating PA317cell linescontaining 3-globinviruses,

we found many lines containing rearranged proviruses

(Table 1), especially with LNB*HP and LNB*SX. These viruses were in general shorter than the parental virus and had suffered various deletions (some complete) of the

P-globin insert. Similar deletions were found in several inde-pendent clones, suggesting thateitheralternativesplicingor recombination in regions ofhomology was responsible. The rearranged viruses werealways ableto replicate toahigher titer than the original virus. For a given vector there was a correlation betweenthetransient titer of virus obtained from *J2 cells (e.g., Table 2), the highest titer that could be obtainedfrom PA317 cellsinfected withthevector(Table 1), and the proportion of PA317 clones analyzed that contained unrearranged proviruses (Table 1). We interpret these

re-sultstomean thatrecombinant viruses aregenerated during

transfection for all viruses, but only if the transfected virus replicatesverypoorlyarethe rearranged virusesdetectedat highfrequency in the infected PA317 cells. Once packaging cell lines containing single unrearrangedviruseswere

gener-ated, little rearrangement was detected when virus from these cells wasused to infect other cells.

The LNB*SA virus developed here canbe obtained from

4j2

cellsat4 x

10'

CFU/ml, and thistiter allowed transfer of

the

P-globin

gene into murine CFU-S cells. Twelve-day CFU-S cells share several characteristics with more

primi-tive hematopoietic stem cells. They are present in bone

marrowatlowabundance, they cycle slowly, and they give

risetocellsofmultiplelineages. Weshowed thatupto100% of 12-day CFU-S cells can be infected and that every

infected CFU-S cell expresses correctly initiated human

,*-globin

mRNA. The high titer and ability ofthis virus to

expressthe 3-globingeneinvivo will assistfurther studies of

the long-term expression of the introduced P-globingene in

transplanted mice and in human hematopoietic cells.

ACKNOWLEDGMENTS

WethankCarol Buttimore fortechnical assistance, Thalia

Papa-yannopoulou for help with the immunofluorescence studies, and

Ronald Reeder forsuggestions concerning the manuscript.

M.A.B.wassupportedbytheMedicalScientist Training Program

ofthe National Institutes of Health. This study was supported by

Public Health Service grants CA41455, HL36444, HL37073, and AG00057 from the NationalInstitutesofHealth.

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ERRATUM

Design of Retrovirus Vectors for Transfer and Expression of the Human

f3-Globin Gene

A. DUSTY MILLER, M. A. BENDER, EDITH A. S. HARRIS, MICHAEL KALEKO AND RICHARD E.GELINAS DepartmentofMolecularMedicine, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104, and

DepartmentofPathology, University of Washington, Seattle, Washington 98195

Volume 62, no. 11, p. 4337: The lastline of the right-hand column should read ". . . in the same volume of fresh medium plus 3 mM HMBA. Cells were harvested on day 6."