RAPID COMMUNICATION

High Levels of Human Glucocerebrosidase Activity in Macrophages of Long-Term Reconstituted Mice After Retroviral Infection

of Hematopoietic Stem Cells

Pamela H. Correll, Susan Colilla, Harish P.G. Dave, and Stefan Karlsson

Gaucher disease is a leading candidate for somatic gene therapy using bone marrow (BM) cells as target tissue. Towards this end, we have constructed a retroviral vector (LG) in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). Day 12 to 14 colony-forming unit- spleen progenitor cells were infected by the LG virus with a 100% efficiency, and GC messenger RNA (mRNA) and protein were detected in the progeny of these cells. Tissues from long-term reconstituted mice analyzed 8 months posttrans- plantation with LG-infected BM contained the intact provirus at greater than 1 copy per cell, indicating effective infection of hematopoietic stem cells. Human GC mRNA generated by the viral LTR was detected in macrophages as well as other

AUCHER DISEASE is an autosomal recessive disor- G der caused by the inherited deficiency of the Iysoso- mal enzyme glucocerebrosidase (GC).’ This deficiency results in the accumulation of the glycolipid glucocerebro- side in bone marrow (BM)-derived macrophages causing hepatosplenomegaly, osteolytic degeneration of the skele- ton, and, in some cases, progressive neurologic deteriora- tion? Enzyme replacement therapy with macrophage- targeted human placental GC has been successful at reversing the signs of Gaucher however, it is not a permanent cure. Allogenic BM transplantation (BMT) has also been beneficial,6f7 but the procedure has a high morbidity and mortality rate and is restricted to patients with matched donors. Gene therapy, transfer of the GC gene into hematopoietic stem cells (HSC) and autologous BMT, may be similarly beneficial without the risk of graft rejection or graft-versus-host disease. In addition, if repop- ulating HSC are transduced, gene therapy would be a permanent cure for Gaucher disease.

Retroviral transfer of the human GC cDNA into hemato- poietic progenitor cells from patients with Gaucher disease resulted in a complete correction of the enzyme defi- ~ i e n c y . ~ ~ ~ In the mouse, the human GC gene has been transfered into day 12 to 14 colony-forming unit-spleen (CFU-S) multipotential progenitor cells and the human GC protein was expressed in the progeny of these cells in vi~o.~OJ~ Transduction of murine HSC as shown by transplan- tation and long-term reconstitution of recipient mice has been shown1*J3; however, production of human GC protein in the macrophages of these mice was not reported. Several other genes have been transferred into murine HSC,14 often with similar problems in obtaining long-term protein produc- tion at high levels from the transferred gene.15

Human GC protein has been shown by immunohisto- chemistry in the nonadherent murine hematopoietic cells of a long-term culture after infection of long-term culture- initiating cells (LTCIC); however, human GC enzyme activity was not reported.1° We now present, for the first

hematopoietic cells. Enzyme activity was increased fivefold and twofold in macrophages from BM and spleen, respec- tively, and could be precipitated with an antibody specific for human GC. Immunohistochemical analysis detected the hu- man GC protein in 81% of the macrophages from five recipient mice. These data indicate that, after transduction of hematopoietic stem cells, the LG vector is capable of direct- ing expression of human GC in the majority of macrophages from long-term reconstituted mice and producing enzyme levels comparable with endogenous mouse activity, suggest- ing that this virus may be useful in the treatment of Gaucher disease. This is a US government work. There are no restrictions on its use.

time, production of human GC protein in a great majority of the macrophages from long-term reconstituted mice transplanted with retrovirally transduced HSC. The levels of human GC enzyme activity in the macrophages are equal to or greater than the endogenous mouse GC activity. The results presented here indicate that therapeutic enzyme levels in these cells have been achieved.

MATERIALS AND METHODS

The LG vector used in this study was derived from the LN series of retroviralvectors.’6 A plasmid (Gl), in which the NeoR gene from LN was removed and replaced with a multiple cloning site, was obtained from Martin Eglitis (Genetic Therapy Inc, Gaithersburg, MD.) The 2.3-kb EcoRI fragment of the human GC cDNAI7 was cloned into the EcoRI site of G1.

Cells and viruses. The LN vector was cotransfected with pSV2Neo into the GP+E86 packaging cell line.’* The cells were selected in 1 g/L G418 and the titer of neo-resistant clones was determined on thymidine kinase-negative 3T3 cells by Southern blot analysis. The clone used in this study had a titer of up to 1.0 copy/cell when compared with copy number controls. This clone was free of helper virus as determined by a marker rescue assay19 using 3T3 cells infected with a retroviral vector containing the NeoR gene.

Retroviral vector.

~

From the Molecular and Medical Genetics Section, Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD.

Submitted March 25,1992; accepted May 12,1992. Supported in part the National Gaucher Foundation by a grant

(NGF 26) to S.R This work was performed in partial fulfillment of the requirements for the Ph.D. degree in genetics at The George Washing- ton Universiv, Washington, D. C. by P. H. C.

Address reprint requests to Stefan Karlsson, MD, PhD, Bldg IO, Rm 3004, National Institutes of Health, Bethesda, MD 20892.

The publication costs of this artid? were defrayed in part by page charge payment. This article must therefore be hereby marked “adver- tisement” in accordance with 18 US. C. section 1734 solely to indicate this fact.

This is a USgovemment work There are no restrictions on its use. 0006-4971 /92/8002-0037$0.00/0

Blood, VOI 80, NO 2 (July 15). 1992: pp 331-336 33 1

332 CORRELL ET AL

Animals. C57BL/J6 mice were used as BM donors and WBB6FI mice were used as BM recipients. All mice were obtained from Jackson Laboratories (Bar Harbor, ME). Recipient mice were irradiated with 850 rads before transplantation.

BM was harvested from donor mice 3 days after intravenous injection with 150 mg/kg 5-fluorouracil. The cells were prestimulated for 2 days in the presence of 200 U/mL recombinant interleukin-3 (IL-3) and IL-6 and with or without 0.1 pg/mL murine mast cell growth factor (MGF)?O and cocultured with the virus producers for 2 days under the same conditions in the presence of 8 pg/mL polybrene. Addition of MGF to the stimulation medium did not seem to affect the overall infection efficiency (unpublished observations, Febru- ary 1992). Recipient mice were injected with 5 x 10" to 1 x 105 cellslmouse for individual CFU-S foci and 1 to 2 x 106 cells/mouse for confluent spleens and long-term reconstituted mice. Tissues were analyzed for the presence of human GC DNA, RNA, and protein on day 13 for CFU-S foci and 8 months posttransplantation for long-term reconstitution. Macrophages isolated from BM and spleen were grown for 8 days as described.12.21

Southern blot analysis was performed using standard techniques. Total cellular RNA was extracted by guanidine thiocyanate22 and separated on a formaldehyde/agarose gel. The RNA was transfered to nitrocellulose filter, prehybridized, hybridized, and washed as described.=

Cell pellets were extracted in a 50 mmol/L potassium citrate/potassium phosphate buffer (pH 5.9) containing Triton X-100 (2 mg/mL) and freeze-thawed for three cycles. Cell extracts were spun for 30 minutes at 12,000 rpm and cleared cellular lysates were assayed for GC activity. GC activity was assayed by cleavage of the synthetic substrate 4-methylumbelliferyl glucopyranoside (Sigma, St Louis, MO) at 4.8 mmol/L in a 0.1 mol/L potassium phosphate buffer (pH 5.9) with 1.5 mg/mL Triton X-100 and 1.25 mg/mL sodium taurocholate at 37°C. The reaction was terminated with 0.4 mol/L NaOH/0.4 mol/L glycine, and cleaved 4-methylumbelliferone was measured using a fluorimeter.

For each sample, two identical tubes were set up containing 10 pL of cleared cellular lysate and 1 pL bovine serum albumin (BSA) (10 mg/mL). Ten microliters of the 8E4 monoclonal antibody (2.5 mg/mL),24 specific for human GC, was added to one tube of each pair. All tubes were brought up to 50 pL total volume with phosphate-buffered saline (PBS) and were rotated at 4°C overnight. Fifty microliters of a 10% cell suspension in PBS of IgG Sorb (The Enzyme Center, Malden, MA) was added to each sample and tubes were rotated at room temperature for 30 minutes. Samples were centrifuged at 12,000 rpm for 3 minutes and two 10-pL aliquots of supernatent were removed for measurement of GC enzymatic activity.

Immunohistochemical analysis. Macrophages isolated from spleen were grown on slides for 8 days. The cells were washed in PBS and k e d in methanol for 20 minutes. Immunochemical detection of human GC protein was performed according to directions using the Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA), which contains a biotinylated goat antimouse IgG and avidin-biotin-peroxidase complex. 8E4 (2.5 mg/mL) at a 1:320 dilution was used as the primary antibody and PBS/O.5% Tween-20 as the dilution buffer. Positive cells were identified by staining with the horseradish peroxidase substrate 3,3'-diaminoben- zidine (DAB). The cells were counterstained with hematoxylin.

Protein was extracted from cells as described above, separated by sodium dodecyl sulfate-polyacryl- amide gel electrophoresis (SDS-PAGE), and transferred onto a nylon membrane by electrical current. Nonspecific protein binding was blocked by agitating the blot for 1 hour in PBS/O.O5% Tween-20 containing 10% dried non-fat milk. The blot was then incubated for 90 minutes with a 1:2,000 dilution of a rabbit

BM infection and transplantation.

DNA and RNA analysis.

GCenzymatic assay.

Immunoprecipitation of human GC.

Westem blot analysis.

polyclonal antibody to GC, which has a much higher affinity for human GC than mouse GC, in PBS/Tween. The blot was washed with 500 mL of PBS/Tween and incubated with a 1:40 dilution of protein A labeled with in PBS/Tween for 90 minutes. After washing in 500 mL of PBS/Tween, the blot was covered with cellophane and dried on a slab gel dryer for 30 minutes under heat. The blot was exposed at -70°C.

RESULTS

The GP+E86/LG packaging cells were assayed for human GC protein production to determine whether the LG vector (Fig 1) is capable of producing functional GC. Western blot analysis showed production of a large amount of the correct size protein in these cells that crossreacted with anti-GC antibody (data not shown). When these cells were analyzed for GC enzyme activity, they were shown to produce 2.65-fold higher levels (specific activity [SA] = 9.4 nmol4-MU/min/mg protein) than the GP+E86 packaging cells alone (SA = 3.5 nmol4-MU/min/mg protein).

BM cells infected with the GP+E86/LG virus were transplanted into lethally irradiated recipient mice and, 13 days later, CFU-S colonies were harvested and tested for the presence of GC DNA, RNA, and protein. Ten colonies were analyzed by Southern blot analysis, and all 10 con- tained the intact LG provirus (100% infection efficiency) (Fig 2A). Northern analysis confirmed that the expected 3.8-kb transcript was being expressed from the viral long terminal repeat (LTR)in six of six colonies tested (Fig 2B). A confluent spleen analyzed by Western blot analysis showed the production of the three normal glycosylated forms (59,62, and 65 Kd) of the human GC protein in these cells (Fig 2C).

Tissues from four long-term reconstituted mice from the same infection were analyzed 8 months posttransplantation with LG-infected BM. Cells from BM and spleen from the four mice were pooled to yield enough macrophages for quantitative analysis of human GC messenger RNA (mRNA) and enzyme activity. Southern blot analysis of DNA from the pooled BM and spleen cells showed the presence of the LG provirus in both tissues at greater than 1 copy/cell when compared with copy number controls (Fig 3A). DNA isolated from thymus of each individual mouse also contained at least 1 to 2 copies/cell, indicating that all four mice were well reconstituted with LG-infected BM stem cells. The high copy number present in these cells indicates that there was a high infection efficiency of HSC, with multiple infections occurring in some of these cells. Whereas integration analysis showed 2 to 6 integration sites/mouse (data not shown), the actual number of trans- duced stem cells cannot be determined. RNA isolated from

I "

GC cDNA H LTR I I

N E E N - 1 Kb

Fig 1. Diagram of the vector used. SD and SA, splice donor and acceptor sites; LTR, MolMLV LTR; (A)n, polyadenylation signal; kb, kilobases; N and E, sites for restriction endonucleases Nhe I and EcoRI.

HUMAN GC ACTIVITY IN MACROPHAGES OF MICE 333

Fig 2. (A) Southern blot anal-

CFU-S foci. DNA was digested with Nhe I (see Fig 1) t o yield a 3.8-kb fragment and 20 pg DNA was loaded per lane. The blot was probed with the human GC cDNA. PC, positive plasmid con- trol mixed with DNA from 3T3 cells; NC, negative control DNA from a CFU-S focus infected with untransfected GP+E86 cells. Sizes are shown in kilobases. (B) Northern blot analysis of six LG- infected CFU-S foci. Twenty mi- crograms of RNA was loaded per lane. The blot was probed with the human GC cDNA. NC, nega- tive control RNA from a CFU-S focus infected with untmmfected GP+E86 cells. Sizes are shown in kilobases. (C) Western blot anal- ysis of a day 13 confluent spleen from a mouse transplanted with LG-infected EM. Three hundred micrograms of total cellular pro- tein was loaded per lane. NC, negative control protein from a confluent spleen infected with untransfected GP+E86 cells. Sizes are shown In kilodattons.

WiS of 10 LG-Infected d q 13

0 0 LG A n z I 1

I

0 LG B z- C

9.5 7.5

4.4

2.4

1.3

-3.8 kb

-110 -84

-47

- 33

nonadherent and adherent (macrophage) cells derived from the spleens of these mice was analyzed by Northern blot analysis (Fig 3B). The results indicate that the viral LTR is directing production of the human GC mRNA in both of these cell fractions, and that expression in macro- phages is at least equivalent to expression in the nonadher- ent cell population.

Protein extracts from macrophages isolated from BM and spleen of the same four long-term reconstituted mice were tested for enzyme activity and compared with protein extracts of macrophages from normal uninfected control mice (Fig 4A). Macrophages from the BM of transplanted mice expressed approximately fivefold higher levels of enzyme activity than the controls and macrophages from the spleen showed roughly a twofold increase in enzyme activity. Immunoprecipitation of the human enzyme with the 8E4 antibody caused a 62.2% and 48.5% reduction in the total activity of macrophages from the BM and spleen, respectively, while causing little or no reduction in activity of macrophages from negative control mice (Fig 4B). This confirms that the increasc in activity seen in these cells is due to the presence of human GC enzyme.

Macrophages were isolated from the pooled spleens of five long-term reconstituted mice from a second experiment performed under similar conditions 8 months after trans- plantation. These macrophages were stained for the pres- ence of human GC protein using immunohistochemistry (Fig 5) . Macrophages from negative control mice did not show any crossreactivity with thc 8E4 antibody, while those from recipients of LG-infected BM stained positively for human GC protein. Three fields of cells were counted under the microscope and in these cells 88 of 109 (81%) of the macrophages were clearly stained positive. This indi- cates that a great majority of the macrophages in these five mice are producing human GC protein.

DISCUSSION

In this study, we have shown that the Moloney murine leukemia virus (MoMLV) viral LTR is capable of driving expression of the human GC gene in macrophages from long-term reconstituted mice after efficient retroviral infec- tion of murine HSC with the LG vector and that the GC protein is being produced in the majority of these cells. The

CORRELL ET AL 334

A

-9.4 -6.6 -4.4

-2.2 -2.0

-1.3

(3 s

-7.5

-4.4

-2.4

-1.3

-0.2 - I-

3.8kb - N I- N-

- 3.8kb

Flg 3. (A) Southem blot anal- ysis of DNA extracted from BM, spleen, and thymus of four mice reconatituted with LG-infected BM 10 months posttransplanta- tion. DNA from EM and spleen was extracted from cells pooled from the four mice. DNA from thymus was extracted from each individual mouse. DNA was cut with Nhe I t o produce a 3.8-kb fragment. The blot was probed with the human GC cDNA. Copy number controls are LG plasmid mixed with 3T3 genomic DNA in the amounts indicated. NC, nega- tive control DNAfrom mouse 3T3 cells. (E) Notthem blot analysis of nonadherent (NA) and macro- phage (Me) fractions isolated from pooled spleens of the four LG-infected long-term mice. GP+E86/LG contains RNA from the packaging cell line used t o infect the EM. NC, negative con- trol RNA isolated from the nonad- herent fraction of spleen from an uninfected mouse. Ten micro- grams of RNA was loaded per lane. Lanes were loaded equally as confirmed by ethidium bro- mide staining of the ribosomal RNA (data not shown). The blot was probed with the human GC cDNA. Sizes are shown in kilo- bases.

levels of enzyme activity seen in the macrophages of these mice is twofold to fivefold higher than the endogenous mouse levels, and 50% or more of that activity is specifically human, as confirmed by immunoprecipitation with the 8E4 antibody. Thc data presented here arc not from one high-expresscr animal; rather, they represent data from pooled cells, serving to emphasize the high transduction efficiency. high GC mRNA production from the LTR, and widespread GC protein production in macrophages.

In previous studies, we have shown efficient infection of murine HSC with retroviral vectors containing the GC C D N A ~ ~ J ~ ; however, neither we nor others” could detect human GC protein in the macrophages of these mice. The main differences between the vector used in present versus earlier studies are the use of the LN vector backbone rather than the N2 vector and the deletion of the neomycin- resistance (NeoR) gene. In the LN vectors, the ATG start codon for translation of the gag protein has been changed

-i A 3s 4

Fig 4. (A) Specific GC enzyme Hthritv (nmol CMU cleavedlminlmg protein) in macrophages derived from the BM and spleen of (m) negative control mice (NC) and (0) mice transplanted with LG-infected BM 10 months posttmnsplantation (LG). (6) Percent of the total activity in (A) precipitated by the 8E4 monoclonal antibodv s~ecific for human GC. This

2 15

1 li . .

represents human GC activity. RO\I \ l \ K K O W 41’1 ! . I . \

HUMAN GC ACTIVITY IN MACROPHAGES OF MICE 335

Fig. 5. Photomicragraph of mnrophages iloloted from the spleen of (A) a negative control mouaa and (B through D) a mouse monrtrtuhd with LO-Infected EM 8 months after transplantation. Cells are stained with the 8E4 monoclonal antlbody speclfk for human GC. All photomicrographs except (D) are taken at a 4OOx magnification; (Dl is taken at 1,OOOx magnification.

to a stop codon and an upstream CT% start codon for the glycosylated gag protein has been removed. This allows for the translation of both spliced and unspliced mRNA transcribed from the LTR,I6 removing the earlier depen- dency of GC cDNA expression upon splicing of the mRNA transcript. The NeoR gene was removed from the vector due to increasing evidence that single-gene vectors often lead to higher levels of expression than double gene vectors after transfer into CFU-S progenitor cells and repopulating stem cell~.1~-=-?~

When the mice in the previous study were examined 4 months posttransplantation, we detected much lower levels of LTR-generated transcript in the macrophages when compared with mRNA levels in nonadherent cells. In the present study, macrophages and nonadherent cells contain roughly equivalent amounts of human GC mRNA gener- ated by the LTR. It is possible that incomplete reconstitu- tion of the tissue macrophage compartment in the W / W recipients used earlier has occurred by 4 months posttrans- plantation and may, therefore, account for the differences in mRNA levels that we observed. By using irradiated

recipients and studying them at 8 months posttransplanta- tion, fuller reconstitution of the macrophages may have occurred, giving rise to higher levels of the human GC transcript. We are currently looking at the timecourse for reconstitution of the macrophage lineage in different tis- sues of the mouse.

This is the first demonstration of expression of the human GC protein in the vast majority of macrophages in long- term reconstituted mice after efficient retroviral-mediated gene transfer of the human GC gene into HSC. The enzyme levels being produced in macrophages from both spleen and BM are at least equal to the endogenous murine levels. This high level of enzyme production from the LG vector in murine macrophages would almost certainly be therapeutic if replicated in patients with Gaucher disease.

ACKNOWLEDGMENT

We thank Dr R.O. Brady for generous support and encourage- ment, Dr M Eglitis for the G1 plasmid, Dr G. Murray for providing antihodies, and Dr David W i n e for reviewing the manuscript.

336 CORRELL ET AL

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