Enhanced retroviral gene transfer into CML and normal bone marrow, and CML and mobilized peripheral...

Enhanced retroviral gene transfer into CML and normal bone

marrow, and CML and mobilized peripheral blood CD34� cells

using the recombinant ®bronectin fragment CH-296

EL AIN E GARRETT, MARINA I. GARIN, ANGELA R-M. MILLE R, JOHN M. GOLDMAN, JUNI A V. MELO AND JA NE F. APPE RL EY

Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London

Received 2 June 1999; accepted for publication 28 July 1999

Summary. Autologous stem cell transplantation is a thera-peutic alternative for many chronic myeloid leukaemia(CML) patients ineligible for the only curative treatment ofallogeneic bone marrow transplantation. In this study theretroviral transduction of CD34� progenitor cells isolatedfrom the bone marrow (BM) and peripheral blood (PB) ofpatients with CML was compared to that of CD34� cellsisolated from the BM and PB of normal individuals andpatients with non-haematological malignancies. A highlysigni®cant increase in transduction of all cell types wasachieved in the presence of the recombinant ®bronectinfragment, CH-296 (P<0´05). In the absence of ®bronectin,centrifugation produced a marginal improvement in thetransduction of all cell types, which was signi®cant only forCMLBM progenitor cells (P <0´05). There was no signi®cant

additive effect when centrifugation was included in the®bronectin infection protocol. In the presence of CH-296,combinations of three or more cytokines improved transduc-tion for all cell types. The same degree of transduction wasobserved for both normal and CML cells, irrespective of thevariations employed in the infection protocol, suggestingthat both leukaemic and non-leukaemic progenitors areequally susceptible to retroviral infection. These resultsdemonstrate that CH-296 has a universal bene®cial effect onthe transduction of haemopoietic progenitor cells, with clearpotential for future clinical trials.

Keywords: chronic myeloid leukaemia, CD34� progenitorcells, retroviral transduction, CH-296, cytokines.

Chronic myeloid leukaemia (CML) is a malignancy of thehaemopoietic stem cell, resulting in abnormal growth anddistribution of clonal progeny. Many CML patients are noteligible for allogeneic bone marrow transplantation, which isat present the only curative treatment for this disease.Autologous stem cell transplantation may offer a therapeuticalternative for these patients (Hoyle et al, 1994). Althoughautografting may be associated with prolonged survival,complete remissions are rare and most patients relapse andprogress to blastic transformation. Relapse may result fromleukaemic cells in the autograft or from residual malignantcells surviving following preparative myeloablative treat-ment for the transplant.

Retroviral gene marking of autologous cells may answermany crucial questions regarding the engraftment potentialof different sources of cells (e.g. bone marrow (BM) versus

peripheral blood (PB)), both normal and leukaemic, andtheir contribution to relapse and haemopoietic recovery. Apioneering study carried out by Deisseroth et al (1994)demonstrated the possibility of using this approach as an invivo model for studying relapse in CML following autograft-ing. It emerged from this work that suf®cient leukaemic cellsare present in the transfused marrow to contribute to relapseand highlighted the need for improved ways of purgingmalignant cells from the autograft. A novel strategy has beenproposed by Zhao et al (1997) using a retroviral vectorcontaining a drug resistance gene, together with BCR-ABLantisense sequences. This protects normal cells from toxicchemotherapy, while abrogating the expression of the BCR-ABL oncogene, which plays a pivotal role in the pathogenesisof this disease (Bhatia & Verfaillie, 1998).

Although these studies have established the feasibility ofusing retroviruses to introduce genes into CML cells, otherobstacles remain which limit its ef®cacy for all cell types.These relate to the mode of retroviral-mediated gene transferand the search for a safe, effective and reliable method for

British Journal of Haematology, 1999, 107, 401±408

401q 1999 Blackwell Science Ltd

Correspondence: Dr Jane F. Apperley, Department of Haematology,Imperial College School of Medicine, Hammersmith Hospital,

DuCane Road, London W12 ONN.

transducing haemopoietic stem cells, typi®ed by the presenceof the CD34 antigen, has led to many new approaches in this®eld. A notable advance in gene transfer technology is therecombinant ®bronectin fragment CH-296, which interactsspeci®cally with the target cell and the retrovirus (Hanen-berg et al, 1996). Activated integrins VLA-4 and VLA-5 onthe surface of the cell, mediate adhesion to the CS1 sequenceand cell binding domain of the ®bronectin molecule; whilethe negatively charged retrovirus interacts via electrostaticforces with the positively charged heparin binding domain of®bronectin (Hanenberg et al, 1996). The increased proximityof the target cell and retrovirus is thought to facilitate virusentry and thus lead to greater gene transfer than has beenachieved in the past (Hanenberg et al, 1997). In recent yearsan increasing number of reports have con®rmed the bene®tsof using full-length human ®bronectin and a variety ofrecombinant human ®bronectin fragments to enhance thetransduction of haemopoietic target cells (Conneally et al,1998; Hanenberg et al, 1996; Moritz et al, 1994, 1996;Ward et al, 1994). However, further studies need to beundertaken to determine its ef®cacy in relation to CML cells,as these cells are characterized by their inability to adhere tostroma and ®bronectin (Dowding et al, 1991; Gordon et al,1987; Verfaillie et al, 1992).

Although the main failing in previous gene transferstudies has been the low level of in vivo gene marking(Deisseroth et al, 1994; Dunbar et al, 1995; Emmons et al,1997), these trials have demonstrated that it is possible totransduce and engraft both normal and malignant haemo-poietic progenitors. In fact there has been some suggestionthat the latter are more susceptible to retroviral transduction(Cornetta et al, 1994). We sought to address this byoptimizing the transduction and culture conditions ofprogenitor cells isolated from the BM and PB of patientswith CML and to compare this to the transduction ef®ciencyof BM and PB from normal individuals or patients with non-haematological malignancies. The primary objective of thisstudy was to develop a reproducible and ef®cient, clinicallyapplicable protocol for transducing these cells. To this end anumber of different infection protocols involving therecombinant ®bronectin fragment CH-296 were employed.A highly signi®cant increase in transduction of all cell typeswas achieved in the presence of ®bronectin, with little effectobserved when the cells were centrifuged with the viralsupernatant. Cocktails of three or more cytokines werebene®cial for all cell types.

MATERIALS AND METHODS

Retroviral vectors. The retroviral vector pBabeNeo,containing the neomycin phosphotransferase (neo) geneexpressed from the SV40 early region promoter(Morgenstern & Land, 1990), was transfected by calciumphosphate precipitation into GP�E86 (Markowitz et al,1988a), an ecotropic packaging cell line. The resultingecotropic producer cell line was cloned by selection in G418(700 mg/ml active concentration; Gibco BRL, LifeTechnologies Ltd, Paisley) and virus supernatant from thehighest-titre clone was used to infect GP�envAM12 cells

(Markowitz et al, 1988b). This amphotropic producer cellline was cloned in turn and supernatant from the highesttitre clone, with a titre of 7 ´ 105 to 1 ´ 106 colony-formingunits (cfu)/ml on NIH3T3 cells, was used in all theexperiments described. Both ecotropic and amphotropicproducer cell lines were negative for replication competentretrovirus when tested on three separate occasions using themarker rescue assay (Markowitz et al, 1988a).

Primary CD34� haemopoietic cells. Normal bone marrow(NBM) was obtained from normal volunteers donating forallogeneic bone marrow transplantation. Mobilized peri-pheral blood stem cells (PBSC) were obtained from patientswith non-myeloid malignancies, e.g. myeloma, breastcancer, amyloidosis, after administration of granulocyte-colony stimulating factor (G-CSF) alone or chemotherapyand G-CSF. These samples represented a source of `normal'myeloid progenitors. Peripheral blood, collected by leuka-pheresis, and bone marrow samples were obtained fromuntreated CML patients in the initial chronic phase of thedisease. Cryopreserved CMLBM samples were also used inthis study when fresh material was not available (4/5samples analysed). All blood and bone marrow specimenswere obtained with informed consent from surplusmaterial collected for diagnostic or therapeutic purposes.Mononuclear cells were isolated by density gradientcentrifugation (Lymphoprep; Nycomed Pharma AS, Oslo,Norway) and then enriched for CD34� cells using theMinimacs system (MACS CD34 Isolation Kit, MiltenyiBiotec Inc., Sunnyvale, Calif.), according to the manufac-turer's recommendations. Samples of CD34� cells withpurities of >90% were routinely obtained following CD34separation (data not shown).

Transduction protocol. In a ®rst series of experiments,freshly isolated CD34� cells were prestimulated overnightwith 50 ng/ml recombinant human IL-3, IL-6, SCF and1 ng/ml IL-1b (Sigma-Aldrich Company Ltd, Poole) in a-MEM media (Gibco BRL), supplemented with 15% fetal calfserum (FCS, Harlan Sera-Lab Ltd, Belton, Loughborough).The next day the cells were transduced on ®bronectin orbovine serum albumin (BSA, Sigma-Aldrich)-coated plates,with cell-free supernatant containing the pBabeNeo retro-virus. On average 2 ´ 105 cells were incubated with 2 ml ofsupernatant, resulting in a multiplicity of infection (MOI) of7±10. For ®bronectin-assisted transduction, non-tissueculture treated plates were coated with 8 mg/cm2 CH-296(RetroNectinTM; kindly donated by Takara Shuzo Co. Ltd,Shiga, Japan). For supernatant infections, plates were coatedwith 2% BSA, and polybrene (Sigma-Aldrich) at 4 mg/ml wasadded to the retroviral supernatant and incubated with thecells overnight. Control cells were mock transduced on BSA-coated plates. In experiments where centrifugation was usedto enhance the transduction ef®ciency, the ®bronectin andBSA-coated plates containing the cells plus retroviral super-natant, were centrifuged (1000 g) for 2 h at 208C and thenincubated at 378C as usual.

In subsequent experiments, to evaluate the effect ofdifferent combinations of growth factors on the transductionef®ciency, cells were prestimulated and transduced on®bronectin-coated dishes, either without growth factors or

q 1999 Blackwell Science Ltd, British Journal of Haematology 107: 401±408

402 Elaine Garrett et al

403CH-296-enhanced Transduction of CML Cells


using the following combinations of cytokines: (a) inter-leukin (IL)-1, IL-3, IL-6, stem cell factor (SCF); (b) IL-1, IL-3,IL-6, SCF, ¯t-3-ligand (FL) (50 ng/ml; First Link UK Ltd,Brierley Hill); (c) IL-3, SCF, FL; (d) IL-6, SCF. When SCF wasused in isolation with IL-6, its concentration was increasedto 100 ng/ml, as used by Hanenberg et al (1996).

After 24 h the non-adherent cells were harvested and theadherent cells were collected using cell dissociation buffer(Gibco BRL). The combined cells were cultured overnight ina-MEM media containing growth factors and 15% FCS. Thecells were then plated in a granulocyte-macrophage colonyforming unit (CFU-GM) progenitor cell assay, in triplicate, inthe presence or absence of G418 (1 mg/ml active concentra-tion). The ef®ciency of gene transfer was assessed on day 14as the percentage of G418 resistant colonies obtainedfrom transduced cells minus the background percentage ofG418-resistant colonies derived from mock-infected cells.

Polymerase chain reaction (PCR) ampli®cation of the NeoR

gene sequence. In order to con®rm the presence of the markergene, colonies formed in G418-supplemented media weretested by nested PCR ampli®cation of NeoR sequences.Individual colonies were plucked from the methylcelluloseculture into 12 ml PBS, boiled for 10 min and placed on ice.5 ml of the boiled lysate were used as DNA template in a 25 ml®rst round PCR set up in duplicate, with primers JNE1� (50-CAA GCG AAA CAT CGC ATC GAG CGA) and JNE2ÿ (50-TATCGC CTT CTT GAC GAG TTC TTC). A 1 ml aliquot of the ®rst-step PCR product was subjected to a second round ofampli®cation in a 20 ml volume with primers NN5� (50-ATGGAA GCC GGT CTT GTC GAT) and NN3ÿ (50-TTC CTC GTGCTT TAC GGT ATC). Composition of the reactions in bothPCR rounds was 1´ Taq polymerase buffer with 1´5 mM

MgCl2 (Boehringer Mannheim Ltd., Lewes), 250 mM eachdATP, dCTP, dGTP, dTTP, 0´25 mM each sense and antisenseprimer, and 0´025 U/ml Taq polymerase (BoehringerMannheim). Thermocycling conditions for the ®rst-stepreaction were 35 cycles of denaturation at 958C for 30 s,annealing at 608C for 50 s and extension at 728C for 1 min,followed by a ®nal 10 min extension at 728C. Thermocyclingconditions for the second-step reaction were 31 cycles ofdenaturation at 958C for 30 s, annealing at 648C for 50 s and

extension at 728C for 1 min, followed by a ®nal 10 minextension at 728C.

5 ml of the second-step products were visualized byelectrophoresis through ethidium bromide stained 2%agarose gels. PCR ampli®cation of an 880 bp genomicfragment of the human ABL gene was performed asdescribed elsewhere (Melo et al, 1994) on negative coloniesto con®rm the presence of ampli®able genomic DNA.Colonies formed by untransduced cells were used as controlsin the PCR reactions together with methylcellulose blanksplucked from these control plates.

Statistics. Statistical analyses were performed using Inter-cooled Stata 5.0 for Windows '95 (Stata Corporation, EastCollege Station, Texas). The medians of two populations werecompared using a non-parametric test, the Wilcoxon rank-sum test.

RESULTS

Effect of recombinant ®bronectin on transductionof CD34� cellsInitial experiments examined the effect of the ®bronectinfragment CH-296 on the transduction ef®ciency of CD34�

cells isolated from PBSC, CMLPB, CMLBM and NBM. Cellswere pre-stimulated overnight with IL-1, IL-3, IL-6, SCF andtransduced in the presence of recombinant ®bronectin orBSA. CH-296 was effective in augmenting the transductionof CD34� cells from each source as shown in Table I. Incontrast the traditional method for infecting cells withretroviral supernatant and polybrene was equally inef®cientfor all cell types.

The transduction of PBSC progenitors increased signi®-cantly (P <0´005) from a median of 0´4% (range 0±6´4%)for supernatant infection on BSA to 7´6% (range 1´1±29´5%) on ®bronectin-coated dishes. A highly signi®cantincrease was also observed for CMLPB (P <0´0001), with amedian transduction ef®ciency of 1´0% (range 0±4´9%)when cells were infected using supernatant versus a medianof 13´0% (range 3´4±28´8%) in the presence of recombinant®bronectin. Similarly for NBM a median transductionef®ciency of 0´6% (range 0±3´9%), was observed with

Table I. Comparison of supernatant infection in the presence and absence of the

recombinant ®bronectin fragment CH-296.

Transduction ef®ciency (%)*

Cell type n Supernatant infection n Fibronectin infection

CMLPB 13 1´0 (0±4´9) 18 13´0 (3´4±28´8)

PBSC 7 0´4 (0±6´4) 14 7´6 (1´1±29´5)

NBM 6 0´6 (0±3´9) 11 10´4 (2´3±38´4)CMLBM 5 1´0 (0±1´3) 6 13´4 (8´2±20´5)

For each cell type comparisons between ®bronectin infection and supernatant

infection yielded statistically signi®cant differences (P <0´05) (Wilcoxon rank-sumtest).

* Results represent the median transduction ef®ciencies of n separate experiments.

supernatant alone, which increased to a median of 10´4%(range 2´3±38´4%) when ®bronectin was added to theprotocol (P <0´05). CMLBM cells were also infected effec-tively in the presence of ®bronectin (13´4%, range 8´2±20´5%), in contrast to the result obtained in its absence(1´0%, range 0±1´3%; P<0´05).

Con®rmation of results using nested PCR for the NeoR geneTable II shows two representative experiments where nestedPCR for the neomycin resistance gene was performed induplicate on G418R CFU-GM colonies from CMLPB cells.Greater than 90% of the colonies evaluated were positive forthe presence of the marker gene. These results con®rm the®ndings of the colony assays where the percentage of thetransduction ef®ciency is calculated according to the numberof G418-resistant colonies obtained.

Effect of centrifugation on transduction of CD34� cellsTo evaluate the putative enhancing effect of centrifugation

on supernatant infection in the presence and absence of®bronectin, a 2 h centrifugation step was included in theprotocol. Table III summarizes the results obtained when thefour different infection protocols were compared. In theabsence of ®bronectin, centrifugation produced a marginalimprovement in the transduction of all cell types shown,which was signi®cant only for CMLBM progenitor cells(P<0´05). For CMLPB, CMLBM, PBSC and NBM the mediantransduction ef®ciency increased from 1´2% (range 0±4´9%), 1´0% (range 0±1´3%), 0´5% (range 0-6´4%) and0´6% (range 0±3´9%) respectively, to 4´1% (range 2´0±13´0%), 3´7% (range 3´4±4´8%), 1´8% (range 0´8±7´9%)and 0´8% (range 0´1±3´9%) when these cells werecentrifuged with the viral supernatant.

In all cases supernatant infection in the presence of®bronectin was superior to supernatant infection withcentrifugation (P<0´05 for each sample type, Table III).The median percentage of transduced cells increased from4´1% to 13´4% for CMLPB cells, from 3´7% to 13´5% for



Table II. Representative results of nested PCR for the neomycin resistance gene.

Total number of No. ofTransduction G418R colonies G418R colonies

ef®ciency (%) tested by nested positive for the

Experiment Method of infection (G418R colonies) PCR* NeoR gene

1 Supernatant 2´3 2 2Supernatant� Fibronectin 10´8 28 28

2 Supernatant 1´7 4 4

Supernatant�Centrifugation 5´6 17 16Supernatant� Fibronectin 7´8 17 17

Supernatant� Fibronectin�Centrifugation 7´8 22 22

* Each colony was analysed in duplicate, together with the appropriate controls (refer to Materials and Methods).

Table III. Effects of different infection methods on the transduction of CD34� cells (%) from different haemopoietic tissues.*

Cell type

Infection method CMLPB PBSC CMLBM NBM

Supernatant 1´2 (0±4´9) [n�5] 0´5 (0±6´4) [n�5] 1´0 (0±1´3) [n�5] 0´6 (0±3´9) [n�5]

Supernatant�

Centrifugation 4´1 (2´0±13´0) [n�5] 1´8 (0´8±7´9) [n�5] 3´7 (3´4±4´8) e [n�5] 0´8 (0´1±3´9) [n�5]Supernatant�

Fibronectin a, c 13´4 (7´8±28´8) [n�5] 8´1 (4´8±20´3) [n�5] 13´5 (8´2±20´5) [n�5] 11´3 (5´7±38´4) [n�7]

Supernatant� Fibronectin�

Centrifugation b 11´1 (7´8±26´2) [n�5] 10´3 (2´9±17´4) [n�5] 10´1 (5´2±14´7) d [n�5] 9´4 (0±43´9) [n�7]

* Results are the median transduction ef®ciency of n separate experiments (range shown in parentheses). For each cell type, infection methods

were compared using the Wilcoxon rank-sum test.a±e Represent comparisons between different infection methods which yielded signi®cant results (P<0´05): a supernatant infection versus

supernatant� ®bronectin, P<0´05 for each cell type; b supernatant infection versus supernatant� ®bronectin� centrifugation, P<0´05 for

each cell type; c supernatant� centrifugation versus supernatant� ®bronectin, P<0´05 for each cell type; d supernatant� centrifugation versus

supernatant� ®bronectin� centrifugation, P<0´05 for CMLBM; e supernatant infection� centrifugation versus supernatant infection, P<0´05for CMLBM.



CMLBM cells, from 1´8% to 8´1% for PBSC cells and from0´8% to 11´3% for NBM cells. This represents a 3-fold, 4-fold,5-fold and 16-fold increase for CMLPB, CMLBM, PBSC andNBM respectively. The large fold increase observed for NBMprogenitor cells re¯ects the fact that neither supernatantinfection on its own, nor supernatant infection with theaddition of a centrifugation step, was effective in transducingthese cells. There was no signi®cant additive effect whencentrifugation was included in the ®bronectin infectionprotocol (Table III). Indeed in most cases (CMLPB, NBM,CMLBM) ®bronectin infection with centrifugation showed atrend towards reduction in the proportion of transducedcells.

Effect of different cytokine combinationson gene transfer ef®ciencyAfter establishing the superiority of transduction in thepresence of ®bronectin, we carried out a number ofexperiments to determine the optimal cytokine combinationfor retroviral-mediated gene transfer into CML and normalprogenitors (Table IV). Cells were prestimulated overnightwith different combinations of IL-1, IL-3, IL-6, SCF and FL,and infected with retroviral supernatant in the presence ofrecombinant ®bronectin. For CMLBM the four- and ®ve-factor combinations of cytokines (IL-1, IL-3, IL-6, SCF andIL-1, IL-3, IL-6, SCF, FL), yielded signi®cantly greater genetransfer than using only two growth factors (IL-6, SCF) ornone (P <0´05). A similar trend was observed for the CMLPBcategory, where the results obtained with the four-factorcombination was signi®cantly better than those using eitherIL-6 and SCF or no growth factors (P <0´05). CMLBM cellswere transduced signi®cantly more ef®ciently than CMLPBcells when the growth factor combination IL-6 and SCF wasused throughout the transduction protocol (P <0´05).

In the case of NBM and PBSC, the highest mediantransduction ef®ciencies were observed with the twocytokine combinations that included FL (9´4% and 9´7%

for NBM; 21´8% and 14´1% for PBSC) although these resultswere not signi®cantly different from the other series ofgrowth factors employed. Transduction of NBM CD34� cellsin the presence of IL-1, IL-3, IL-6 and SCF, was signi®cantlygreater than transduction in the absence of any growthfactors (P<0´05). The transduction ef®ciency of PBSC wasnot signi®cantly increased using any one of the differentcytokine combinations.

DISCUSSION

CML is a lethal haemopoietic malignancy for many patientsineligible to receive the only curative treatment of allogeneicbone marrow transplantation. Autologous stem cell trans-plantation may offer a short respite from the disease, butultimately all relapse and progress to blast transformation.Retroviral-mediated gene marking offers some potentialinsights into the biology of this relapse, with the elusivegoal of gene therapy that may eventually result in a cure forthis insidious disease. Considerable progress is being made inthis direction at the molecular level, with the design ofretroviral vectors containing genes that speci®cally targetthe leukaemic cell (Zhao et al, 1997). However, clinical dataare scarce, and at present traditional autologous stem celltransplantation remains the most practical option forclinicians attempting to treat this disease, when there is noalternative therapy.

CML cells are characterized at the molecular level by thepresence of a unique BCR-ABL hybrid gene, which at thecellular level results in a clonal leukaemia of maturegranulocytes. Previously our group has examined thekinetics of proliferation and differentiation in CML-derivedCD34� cells and compared this to normal CD34� cells (Garinet al, 1996). It was observed that culturing CML and normalCD34� cells in the presence of IL-1, IL-3, IL-6 and SCF resultsin similar proliferation of both types of cells. However, theproportion of CML cells co-expressing CD33 (a marker of

Table IV. In¯uence of cytokine combinations on gene transfer ef®ciency (%)*.

Cell type

Infection method CMLPB PBSC CMLBM NBM

IL-1, IL-3, IL-6, SCF a, b 13´4 (4´9±28´8) [n�6] 11´6 (6´3±29´5) [n�5] 13´1 (8´2±16´7) [n�4] 6´4 (5´5±10´4) [n�4]

IL-1, IL-3, IL-6, SCF, FL 11´5 (7´7±12´7) [n�3] 14´1 (5´4±31´6) [n�4] 13´1 (9´0±15´8) c, d [n�4] 9´4 (6´4±16´5) [n�3]IL-3, SCF, FL 7´8 (4´2±23´7) e, f [n�4] 21´8 (8´3±38´2) [n�3] 8´3 (3´6±8´1) [n�4] 9´7 (8´6±17´2) [n�3]

IL-6, SCF 3´5 (1´7±4´0) [n�3] 9´5 (0±29´4) [n�5] 6´8 (4´5±8´1) [n�4] 4´6 (2´4±26´9) [n�3]

No growth factors 0 (0±1´1) [n�3] 4´9 (0±9´7) [n�4] 2´6 (0±4´7) [n�3] 1´4 (0±1´7) [n�3]

* Results are the median transduction ef®ciency of n separate experiments and the range is shown in parentheses. For each cell type, infections

using different cytokines were compared using the Wilcoxon rank-sum test. For NBM and CMLPB transduction in the absence of growth factors,

and the IL-6, SCF combination for CMLPB could be directly compared only with the IL-1, IL-3, IL-6, SCF combination.a±d Represent comparisons between supernatant infection� ®bronectin in the presence of different combinations of cytokines, which yielded

signi®cant results (P <0´05) for each cell type: a IL-6, SCF versus IL-1, -3, -6, SCF, P<0´05 for CMLPB and CMLBM; bno growth factors versus

IL-1, -3, -6, SCF, P<0´05 for CMLPB, CMLBM and NBM; cIL-6, SCF versus IL-1, -3, -6, SCF, FL, P<0´05 for CMLBM; dno growth factors versus

IL-1, -3, -6, SCF, FL, P<0´05 for CMLBM.

The same cytokine combinations were used for both the prestimulation and the transduction periods.

myeloid differentiation) and CD34 at day 0 was much higherthan that of normal cells, indicating that it may be moredif®cult to achieve transduction of very early progenitors inthis malignancy. We concluded from these studies that therewas a window of 3 d during which time CML cells retainedsome of their CD34-positivity and our transduction protocolwas designed accordingly.

In the present study a number of different infectionprotocols were employed to transduce CD34� progenitorcells isolated from the BM and PB of patients with CML, andthese were compared to the transduction ef®ciencies of BMand PB CD34� cells from normal individuals or patients withnon-haematological malignancies. Comparisons betweeninfection with cell-free retroviral supernatant in the presenceand absence of CH-296 demonstrated a highly signi®cantincrease in the transduction of all cell types in the presence of®bronectin. The bene®cial effect of ®bronectin on thetransduction of normal progenitor cells has been demon-strated previously (Hanenberg et al, 1996, 1997). Ourresults with PBSC are comparable to those of Kuga et al(1997) and Breems et al (1998). To our knowledge, only oneother study has examined the effect of ®bronectin on thetransduction of CML CD34� cells, where improved genetransfer was observed using the recombinant ®bronectinfragment FN30/35 (Traycoff et al, 1997). The range oftransduction observed in our study, using CH-296 totransduce CML cells, is comparable to that reported for theFN30/35 fragment.

The increased transduction ef®ciency of CML cells when®bronectin-coated dishes are employed, is perhaps surprisinggiven that CML cells are known to adhere poorly to stromaand more speci®cally to ®bronectin (Dowding et al, 1991;Gordon et al, 1987; Verfaillie et al, 1992). Since CMLprogenitors express similar numbers of integrin adhesionreceptors as normal progenitors, it has been suggested thatthe function of these receptors may be impaired in CML(Verfaillie et al, 1992). This area has become controversial asa recent publication has shown that certain murine cell linestransfected with BCR-ABL display enhanced af®nity for full-length ®bronectin compared with the parental untransfectedcell lines (Bazzoni et al, 1996). Their results were alsoextended to primary CML CD34� cells demonstratingsigni®cantly increased adhesion to ®bronectin as comparedto normal cells. To investigate this further we carried outadhesion assays to detect any CML or normal cells adheringto ®bronectin, in the presence or absence of growth factorsand for varying periods of exposure to CH-296. The numbersof adherent normal and CML cells were very low irrespectiveof the assay conditions and the transduction ef®ciencyobtained (data not shown). Paradoxically, Breems et al(1998) reported a very high af®nity of mobilized CD34�

progenitors to CH-296 (>90% CFC were adherent), but onlyachieved a maximum transduction ef®ciency of 9´5%.Furthermore, a recent study (Yokota et al, 1998) suggeststhat ®bronectin may act as a growth-supporting factor invitro. No such effect was observed in our study when thenumber of CFU-GM colonies obtained in the presence andabsence of ®bronectin were compared (data not shown), inagreement with Moritz et al (1996).

Centrifugation improves transduction of target cells byincreasing the local concentration of retrovirus, therebyaugmenting the rate of virus±cell association (Kotani et al,1994). In our study, centrifugation was carried out in ¯at-bottomed 24-well plates to allow direct comparisons ofcentrifugation in the presence and absence of CH-296.Centrifugation improved the transduction of all cell types,most signi®cantly for CMLBM cells (P<0´05), with the leasteffect observed for NBM (Table III). In most cases centrifuga-tion did not add to the bene®cial effect of ®bronectin. Anumber of studies have evaluated the effect of cytokines onretroviral gene transfer at the in vitro and in vivo level(Brenner et al, 1993a, b; Dao et al, 1997; Emmons et al,1997; Hanenberg et al, 1997). In our experiments withCMLBM and CMLPB the cocktail of four or more growthfactors resulted in signi®cantly greater gene transfer onrecombinant ®bronectin than other combinations tested.A similar trend was observed for NBM and PBSC, althoughthese results were not statistically signi®cant. Studiesby Levesque et al (1995, 1996) have shown that combina-tions of cytokines that synergize to stimulate normal CD34�

cells to proliferate also increase their adhesiveness to®bronectin.

Statistical analysis comparing the transduction of CML PBand BM cells versus their normal counterparts failed to showany signi®cant difference, irrespective of the method orculture conditions employed in the infection protocol. Ourstudy is in contrast to a previous study by Cornetta et al(1994) that examined the transduction of CML cells frompatients undergoing treatment, and achieved much highertransduction of these cells compared to normal cells.Although a recent report has demonstrated enhancedcycling of CML cells compared to normal in the presence ofexogenous cytokines (Traycoff et al, 1998), cell-cycle statusalone is not an accurate predictor of successful gene transfer(Knaan-Shanzer et al, 1996). Our ®ndings suggest thatprotocols optimized for the transduction of normal haemo-poietic cells may be applied to CML cells with similar results.This has important implications for clinical studies aiming atconsistently high levels of gene transfer to leukaemic cells(e.g. autograft purging strategies).

In conclusion, it is clear from this analysis that therecombinant ®bronectin fragment CH-296, has a universalbene®cial effect on the transduction of haemopoieticprogenitor cells. Although the exact mechanism of enhance-ment of gene transfer by recombinant ®bronectin remains tobe fully elucidated, it is evident that this system has a clearpotential in the clinical setting for future trials aimed at genemarking or therapeutic intervention. Moreover it provides asafe effective alternative to more traditional methods such asco-culturing the target cells on retroviral producer cell linesor supernatant infection in contact with stromal cells (Mooreet al, 1992).

ACKNOWLEDGMENTS

The authors thank Richard Syzdlo for advice on statisticalanalyses of the data. This research was supported by a grantfrom the Kay Kendall Leukaemia Fund.





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