TRIM5α Variations Influence Transduction Efficiency With Lentiviral Vectors in Both Human and Rhesus CD34+ Cells In Vitro and In Vivo

Vector immunogenicity, toxicology and safety

original article

© The American Society of Gene & Cell Therapy

TRIM5α Variations Influence Transduction Efficiency With Lentiviral Vectors in Both Human and Rhesus CD34+ Cells In Vitro and In Vivo Molly E Evans1, Chutima Kumkhaek1, Matthew M Hsieh1, Robert E Donahue2, John F Tisdale1 and Naoya Uchida1 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes/National Institute of Diabetes and Digestive and Kidney ­ iseases, National Institutes of Health, Bethesda, Maryland, USA; 2Hematology Branch, National Heart Lung and Blood Institutes, National Institutes D of Health, Rockville, Maryland, USA

Human immunodeficiency virus type 1 (HIV-1) vectors can transduce human hematopoietic stem cells (HSC), but transduction efficiency varies among individuals. The innate immune factor tripartite motif-containing protein 5α (TRIM5α) plays an important role for restriction of retroviral infection. In this study, we examined whether TRIM5α could account for variations in transduction efficiency using both an established rhesus gene therapy model and human CD34+ cell culture. Evaluation of TRIM5α genotypes (Mamu-1, -2, -3, -4, -5, and TrimCyp) in 16 rhesus macaques that were transplanted with transduced CD34+ cells showed a significant correlation between TRIM5α Mamu-4 and high gene marking in both lymphocytes and granulocytes 6 months after transplantation. Since significant human TRIM5α coding polymorphisms were not known, we evaluated TRIM5α expression levels in human CD34+ cells from 14 donors. Three days after HIV-1 vector transduction, measured transduction efficiency varied significantly among donors and was negatively correlated with TRIM5α expression levels. In summary, transduction efficiency in both rhesus and human CD34+ cells was influenced by TRIM5α variations (genotypes and expression levels). Our findings are important for both understanding and mitigating the variability of transduction efficiency for rhesus and human CD34+ cells. Received 18 April 2013; accepted 17 October 2013; advance online publication 10 December 2013. doi:10.1038/mt.2013.256

INTRODUCTION

Though hematopoietic stem cell (HSC)-targeted gene therapy has been proven efficacious in several gene therapy trials,1–7 improvement of transduction efficiency for HSCs is still crucial for further development of gene therapy disorders such as thalassemia and sickle cell disease.8,9 The variability of transduction efficiency for human HSCs also limits development of gene therapy, as unexpectedly low transduction efficiency in HSCs can lead to insufficient therapeutic effects for gene therapy patients. Therefore, we

sought to investigate the cause of the variability in transduction efficiency for human HSCs. A significant restriction factor in retroviral infection is the innate immune factor tripartite motif-containing protein 5α (TRIM5α).10,11 TRIM5α recognizes retroviral capsids in combination with cyclophilin A (CypA) to degrade retrovirus in a species-specific manner.12 In retroviral infection in rhesus macaques, rhesus TRIM5α recognizes the human immunodeficiency virus type 1 (HIV-1) capsid to degrade HIV-1, while the simian immunodeficiency virus (SIV) capsid can escape from rhesus TRIM5α restriction by attaching to rhesus CypA. We previously developed chimeric HIV-1-based lentiviral vectors (χHIV vectors) in which the HIV-1 vector genome is packaged in the context of the SIV capsid permitting escape from rhesus TRIM5α restriction.13,14 The χHIV vector system allows for more efficient transduction of rhesus hematopoietic repopulating cells, compared to the HIV-1 vector; however, transduction efficiency still remains highly variable among animals.13–15 Recently, rhesus TRIM5α polymorphisms have been reported, and rhesus TRIM5α genotype was shown to affect SIV infectivity in rhesus hematopoietic cells.16–21 We hypothesized that TRIM5α variations might influence the variability of transduction efficiency for HSCs with lentiviral vectors. Although several polymorphisms in human TRIM5α have been reported, functional polymorphisms in human TRIM5α occur at a low frequency in the population (1–5%) and are thus not sufficient to account for the variability of HIV-1 infectivity in human cells.22,23 We have previously demonstrated large variability in transduction efficiency for human CD34+ cells with lentiviral vectors.15 The χHIV vector (including the SIV capsid) was observed to have relatively low variability in transduction efficiency for human CD34+ cells compared to the HIV-1 vector. Interestingly, an inhibitor of CypA, cyclosporine, decreased the variability of transduction efficiency with the HIV-1 vector for human CD34+ cells. These data further support our hypothesis that human innate immune factors including TRIM5α and CypA might influence the variability of lentiviral vector transduction efficiency in human CD34+ cells. In this study, we further examined whether the innate immune factors TRIM5α and CypA are

Correspondence: Naoya Uchida, Staff Scientist, Molecular and Clinical Hematology Branch, 9000 Rockville Pike, Bldg 10, 9N112, Bethesda, Maryland 20892, USA. E-mail: [email protected]

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a

HIV-1 vector

χHIV vector

SIV vector

HIV-1 genome

HIV-1 genome

SIV genome

HIV-1 capsid

SIV capsid

SIV capsid

VSVG envelope

VSVG envelope

VSVG envelope

b

Mamu-1

100

Mamu-4

100 80

80

60

60

60

40

40

40

20

20

20

0

0

0.5

1

2

5

Mamu-2

80

60

60

40

40

20

20 0.5

1

2

5

Mamu-3

100

0

80

60

60

40

40

20

20

0

0.5

1

2

0.5

5

0

2

5

0

0.5

1

2

5

1

2

5

TrimCyp

100

80

1

Mamu-5

100

80

0

0.5

Control

100

80

100

%GFP

TRIM5α Effects for CD34+ Cell Transduction

HIV-1 χHIV SIV 0.5

1

2

5

MOI

Figure 1  The χHIV vector escaped from restriction of rhesus TRIM5α Mamu-4 and -5 and TrimCyp. (a) For efficient transduction of rhesus CD34+ cells, we constructed a chimeric HIV-1 vector (χHIV vector) system in which the HIV-1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid to circumvent a species-specific restriction to HIV-1 based vectors. (b) We transduced stable cell lines expressing six different rhesus TRIM5α genotypes (Mamu-1, -2, -3, -4, -5, and TrimCyp) with GFP-expressing HIV-1, χHIV, and SIV vectors. Among all TRIM5α cell lines, transduction efficiency (%GFP) from the χHIV vector fell between that of both the HIV-1 vector and that of the SIV vector. For the χHIV and SIV vectors, %GFP was reduced in Mamu-1, -2, and -3 (P < 0.01) expressing cell lines when compared to that of control cells. For the HIV-1 vector, there was a reduction in %GFP among all TRIM5α types (P < 0.01).

responsible for variability in transduction efficiency with lentiviral vectors in human and rhesus CD34+ cells.

RESULTS Rhesus TRIM5α variations influence lenvitiral vector transduction efficiency in stable cell lines

To evaluate whether rhesus TRIM5α variations influence the transduction efficiency with lentiviral vectors, we transduced cell lines expressing six different rhesus TRIM5α genotypes (Mamu1, -2, -3, -4, -5, and TRIM5α-CypA chimera (TrimCyp)) (Table 1) with enhanced green fluorescent protein (GFP)-expressing HIV1, χHIV, and SIV vectors at multiplicity of infection (MOIs) 0.5, 1, 2, and 5 (Figure 1a). Transduction efficiency was evaluated by GFP-positive frequency (%GFP) in flow cytometry. Among all TRIM5α cell lines, %GFP from the χHIV vector fell between that of the HIV-1 vector and that of the SIV vector (Figure 1b). For the χHIV and SIV vectors, %GFP was reduced in Mamu-1, -2, and -3 Molecular Therapy  vol. 22 no. 2 feb. 2014

Table 1  Rhesus TRIM5α genotypes20 Amino acid position in TRIM5α protein TRIM5α type

307

327

333

339–340

TFP

Mamu-1 or -2

T

P

A

TFP

TFP

Mamu-3

P

P

A

TFP

Q

Mamu-4

P

P

S

Q

Q

Mamu-5

P

T

S

Q

TrimCyp

TrimCyp

P

CypA

TrimCyp

expressing cell lines (P < 0.01 at all MOIs), but not in Mamu-4, -5, and TrimCyp expressing cell lines (at all MOIs except MOI 0.5), when compared to that of control cells. Conversely, the HIV-1 vector revealed a reduction in %GFP among all TRIM5α types (P < 0.01 at all MOIs except TrimCyp at MOI 5). These results 349

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TRIM5α Effects for CD34+ Cell Transduction

Rhesus TRIM5α variations influence transduction efficiency for hematopoietic repopulating cells in rhesus transplantation We evaluated the frequency of rhesus TRIM5α allele types in our rhesus colony at the National Institutes of Health Animal Center (Dickerson, MD), which revealed TRIM5α frequencies of type TFP (Mamu-1, -2, and -3) 46.7%, Q (Mamu-4 and -5) 41.4%, and TrimCyp 11.8% (N = 76; Tables 1 and 2). In addition, evaluation of a colony at the Oregon National Primate Research Center showed frequencies of TFP 35.4%, Q 50.0%, and TrimCyp 14.6% (N = 24). The rhesus colonies at both National Institutes of Health Animal Center and Oregon National Primate Research Center (Beaverton, OR) have a slightly higher frequency of type Q compared to another reported colony (New England Primate Research Center, Southborough, MA);20 however, all rhesus colonies demonstrate similarly high frequencies of TFP and Q compared to the frequency of TrimCyp.20,21 To evaluate whether rhesus TRIM5α genotypes influence lentiviral transduction for rhesus hematopoietic repopulating cells, we analyzed 16 rhesus macaques that were transplanted with CD34+ cells transduced with GFP− (or enhanced yellow fluorescent protein (YFP−)) expressing χHIV vectors (Figure 2a). We evaluated GFP− (or YFP−) positive frequency (%GFP (or YFP)) in granulocytes and lymphocytes 6 months after transplantation, since %GFP plateaus in the peripheral blood at 6 months.13,14,24,25 The %GFP in transduced rhesus CD34+ cells was also evaluated in vitro at the time of transplantation. When we evaluated average %GFP among allele types of rhesus TRIM5α, the TRIM5α type Mamu-4 showed a tendency of higher %GFP both in vitro (CD34+ cells) and in vivo (granulocytes and lymphocytes; Figure 2b); however, we did not observe a significant difference by one-way analysis of variance. For in vivo data analysis (granulocytes and lymphocytes), we excluded three animals (#3:Mamu-3/-3, #15:Mamu-4/TrimCyp, and #16:Mamu-3/-4) that had no engraftment of gene-modified cells, while in vitro %GFP was positive (36–86%) in the transduced CD34+ cells (Table 3). For further statistical analysis, we assessed factors that potentially affect transduction efficiency, including age, sex, weight, total number of mobilized CD34+ cells, cytokine mobilization regimen (granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) (G+SCF) versus G-CSF and plerixafor (G+PL)), cell density during transduction, and TRIM5α genotypes (Table 3). Multivariate analysis demonstrated that TRIM5α type Mamu-4 Table 2  Frequency of rhesus TRIM5α allele in a rhesus colony in the National Institutes of Health Animal Center TRIM5α allele

NIHAC (N = 76)

ONPRC (N = 24)

NEPRC20

TFP/TFP

24%

13%

46%

TFP/Q

34%

38%

36%

TFP/TrimCyp

12%

8%

5%

Q/Q

18%

21%

10%

Q/TrimCyp

12%

21%

1%

0%

0%

2%

TrimCyp/TrimCyp

NEPRC, New England Primate Research Center; NIHAC, National Institutes of Health Animal Center; ONPRC, Oregon National Primate Research Center.

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(Mamu-4 (+): 50.9 ± 19.0% versus Mamu-4 (−): 26.6 ± 16.7%, P = 0.04) as well as mobilization regimen (G+SCF: 48.5 ± 17.4% versus G+PL: 12.7 ± 7.1%, P = 0.01) affected %GFP in CD34+ cells in vitro (Table 4). However, in vivo, only TRIM5α Mamu-4 showed significant effects on higher %GFP among lymphocytes (Mamu-4 (+): 23.7 ± 17.9% versus Mamu-4 (−): 5.3 ± 3.1%, P = 0.046). When analyzing the %GFP among granulocytes in vivo, there was a significant correlation with weight (P = 0.01), mobilized CD34+ cell number (P = 0.02), TRIM5α type Mamu-4 (Mamu-4 (+): 29.3 ± 25.4% versus Mamu-4 (−): 8.4 ± 6.0%, P = 0.03), and mobilization regimen (G+SCF: 25.4 ± 24.1% versus G+PL: ­ 7.5 ± 3.0%, P = 0.04). If in vitro %GFP is included in the analysis, %GFP among granulocytes and lymphocytes are strongly affected by in vitro %GFP (<0.001), and both TRIM5α type Mamu-4 and mobilization regimen are no longer significant. Univariate analysis showed similar tendencies regarding %GFP among granulocytes and lymphocytes (Table 5). Taken together, our data suggest

a

CD34+ cells

In vitro culture

χHIV vector

Transduction (MOI = 50)

CD34+ selection Mobilized PBSCs

Total body irradiation

Rhesus macaques

Transplantation

b 80

%GFP (or YFP)

suggest that both χHIV and SIV vectors can escape from restriction by rhesus TRIM5α Mamu-4, -5, and TrimCyp.

In vitro Granulocytes* Lymphocytes*

60

40

20

0

Mamu Mamu Mamu –3/–4 –1 or 2/–5 –3/–3 n = 5 (4**) n = 5 (4**) n=1 *Twofold scores were used in competitive assay

Mamu –4/–4 n=3

Mamu –4/–5 n=1

Mamu-4/ TrimCyp n = 1 (0**)

**For lymphocytes and granulocytes

Figure 2 Effects of TRIM5α variations on transduction for rhesus CD34+ cells. (a) We analyzed 16 rhesus macaques. The mobilized rhesus CD34+ cells were transduced with green fluorescent protein (GFP) (or yellow fluorescent protein (YFP)) expressing χHIV vectors at multiplicity of infection (MOI) 50. The cells were infused into rhesus macaques following two sequential days of 5Gy (5Gyx2) of total body irradiation for a total dose of 10Gy. We evaluated transduction efficiency (%GFP (or YFP)) in granulocytes and lymphocytes 6 months after transplantation by flow cytometry. %GFP for rhesus CD34+ cells was also evaluated in vitro at the time of transplantation. (b) The TRIM5α type Mamu-4 has a tendency of higher transduction efficiency in rhesus CD34+ cells in vitro, granulocytes, and lymphocytes. For in vivo data analysis (granulocytes and lymphocytes), we excluded three animals, which had no engraftment of gene-modified cells.

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TRIM5α Effects for CD34+ Cell Transduction

Table 3  Background and transduction efficiency in transplanted rhesus macaques CD34+ ID

Age

Sex

Weight (kg)

cell (10e6/kg)

Mobilization

TRIM5α

Cell density (10e5/ml)

%GFP In vitro

Lymphocytesa

Granulocytesa

1

7.3

M

7.92

3.91

G+SCF

Mamu-3/-4

3.50

61.05

40.26

73.42

2

8.4

F

6.36

3.93

G+SCF

Mamu-4/-4

5.10

48.61

24.78b

29.78b <1.00

3

6.6

M

7.86

6.31

G+SCF

Mamu-3/-3

4.96

47.69

<1.00

4

3.9

F

4.62

24.89

G+PL

Mamu-3/-3

7.68

6.96

4.29

8.44

5

4.3

F

4.74

10.34

G+SCF

Mamu-3/-4

4.96

67.20

44.82

37.20

6

4.1

F

5.02

17.73

G+PL

7

3.9

M

5.10

4.43

G+SCF

Mamu-1or2/-5

4.44

10.58

2.43

4.12

Mamu-4/-5

2.26

41.86

14.72

19.90 56.32

8

5.1

M

5.58

8.06

G+SCF

Mamu-4/-4

4.64

64.95

46.18

9

4.5

M

4.78

11.30

G+PL

Mamu-3/-4

5.40

20.58

10.62

9.90

10

4.4

M

5.24

3.63

G+SCF

Mamu-4/-4

3.80

45.81

3.46

2.08

11

3.3

F

4.96

5.65

G+SCF

Mamu-3/-4

5.60

37.45

4.92

5.48

12

2.5

F

4.80

4.58

G+SCF

Mamu-3/-3

4.40

43.68

7.46

6.80

13

5.0

M

6.42

2.96

G+SCF

Mamu-3/-3

3.80

27.30

2.70

4.16

14

3.6

M

4.34

16.13

G+SCF

Mamu-3/-3

7.00

23.09

9.38

18.72

15

2.3

M

4.28

10.28

G+SCF

Mamu-4 /TrimCyp

4.40

86.03

<1.00

<1.00

16

3.5

M

5.39

5.45

G+SCF

Mamu-3/-4

5.88

35.93

<1.00

<1.00

Twofold scores were used in competitive assay. bData for 3 months after transplantation.

a

Table 4  The P values in multivariate analysis regarding transduction efficiency (in vitro, lymphcoytes, and granulocytes) in transplanted rhesus macaques

Table 5  The P values in univariate analysis regarding transduction efficiency (lymphcoytes and granulocytes) in transplanted rhesus macaques

Variables

Variables

In vitro

Lymphocytes

Granulocytes

Lymphocytes

Granulocytes

Age

0.95

0.03

0.28

0.87

0.65

Age

0.09 (R2 = 0.24)

0.03 (R2 = 0.36)

Sex (male versus female)

0.92

0.30

0.95

0.12

0.73

Sex (male versus female)

0.73 (R2 = 0.00)

0.40 (R2 = 0.00)

Weight (kg)

0.85

0.57

0.33

0.04

0.01

Weight (kg)

0.17 (R2 = 0.16)

0.03 (R2 = 0.38)

Mobilized CD34+ cell number

2

0.41

0.001

0.73

0.002

0.02

Mobilized CD34+ cell number

0.50 (R = 0.00)

0.52 (R2 = 0.00)

Cell density during transduction

2

0.55 (R = 0.00)

0.50 (R2 = 0.00)

Cell density during transduction

0.55

0.44

0.91

0.93

0.47

TRIM5α Mamu-4 (0 versus 1 and 2)

0.046 (R2 = 0.32)

0.10 (R2 = 0.22)

TRIM5α Mamu-4 (0 versus 1 and 2)

0.04

0.90

0.046

0.79

0.03

TRIM5α Mamu-4 (1 versus 2)

0.88 (R2 = 0.32)

0.99 (R2 = 0.22)

0.21 (R2 = 0.14)

0.24 (R2 = 0.12)

TRIM5α Mamu-4 (1 versus 2)

0.92

0.33

0.88

0.50

0.70

Mobilization (G+SCF versus G+PL)

Mobilization (G+SCF versus G+PL)

0.01

0.40

0.44

0.97

0.04

—

<0.001

—

<0.001

—

0.61

0.91

0.32

0.92

0.76

%GFP in vitro R2

that TRIM5α type Mamu-4, mobilization regimen (G+SCF), and CD34+ cell transduction efficiency in vitro are important factors that can predict higher %GFP in granulocytes and lymphocytes 6 months following transplantation in rhesus macaques.

Human TRIM5α polymorphisms in the promoter region modestly affect the protein levels and lentiviral transduction in human CD34+ cells We then sought to evaluate whether a human TRIM5α polymorphism could account for the variability of lentiviral vector Molecular Therapy  vol. 22 no. 2 feb. 2014

%GFP in vitro

<0.001 (R2 = 0.67)

0.01 (R2 = 0.50)

transduction efficiency for human hematopoietic cells. However, no common human TRIM5α polymorphism in the coding region has been reported, and yet variability in transduction efficiency for human CD34+ cells is still observed. Therefore, we evaluated polymorphisms in the promoter region (~1k bases) upstream of TRIM5α gene using 14 donors of CD34+ cells, and we recognized three polymorphisms (G-184A, C-737T, and A-945G). Both G-184A and C-737T resulted in relatively lower %GFP and higher TRIM5α protein levels (P < 0.05, TRIM5α protein levels in G-184A), while A-945G has little effect on %GFP and TRIM5α protein levels (Supplementary Figure S1). We also evaluated polymorphisms of human CypA, which is an important innate immune factor for retroviral defense that supports TRIM5α function.26,27 We sequenced known CypA polymorphisms in coding and promoter regions, and two polymorphisms in the promoter 351

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TRIM5α Effects for CD34+ Cell Transduction

region were detected (C1604G and A1650G).26,27 However, these polymorphisms did not affect either %GFP or CypA RNA levels in human CD34+ cells (Supplementary Figure S2).

Human TRIM5α expression levels were negatively correlated with transduction efficiency in human T-lymphocyte cell lines Therefore, we evaluated human TRIM5α RNA and protein levels to determine whether variation in expression of TRIM5α could account for the variability in transduction efficiency. Five human T-lymphocyte cell lines (CEM, CEMx174, A-3, D1.1, and E6-1) were transduced with a GFP-expressing HIV-1-based lentiviral

a

vector at MOIs 0.2, 0.5, and 1.0 (Figure 3a). TRIM5α and CypA RNA levels at the time of transduction were evaluated by reverse transcription real-time polymerase chain reaction, and TRIM5α protein levels were evaluated by Western blot analysis. The transduction efficiency was evaluated by %GFP in flow cytometry 3 days after transduction. Additionally, cell proliferation was evaluated by fold expansion of cell counts during the 3-day culture. All T-lymphocyte cell lines showed an increase of %GFP by MOI escalation, while a large variability of %GFP was observed among cell lines (21.3–69.4% at MOI 0.5; Supplementary Figure  S3). TRIM5α RNA levels were vastly different among the cell lines (815-fold magnitude), while CypA RNA levels were

b T-cell cell lines HIV-1 vector Transduction (MOI = 0.2, 0.5, or 1.0) 2 days

RT-qPCR Western blot

Cell counts flow cytometry

TRIM5α and CypA levels

Fold expansion and %GFP

0

100

80

80

60

60

y = 72.6–11.7x R 2 = 0.828* y = 54.5–10.7x R 2 = 0.722

20 0

y = 35.9–7.68x R 2 = 0.594

0

1

2

3

0

2

4 6 8 10 Cell counts (fold expansion)

12

y = 84.5–34.9x R 2 = 0.764 y = 67.4–34.9x R 2 = 0.804*

20

y = 46.6–27.3x R 2 = 0.782*

0

0.2

0.4

0.6

0.8

1.0

1.2

Relative TRIM5α protein levels

100

MOI 1.0 MOI 0.5 MOI 0.2

80

%GFP

y = 1.39 + 4.14x R 2 = 0.865*

40

0

4

Relative TRIM5α RNA levels

d

40

100

40

y = 8.74 + 5.42x R 2 = 0.932**

60

20

%GFP

%GFP

c

Medium change

y = 24.5 + 5.62x R 2 = 0.949**

80

%GFP

1 day

100

*P < 0.05, **P < 0.01

60 40 20 0

0

0.5 1.0 1.5 Relative CypA RNA levels

2.0

Figure 3  Negative effects of TRIM5α levels on transduction for human T-lymphocyte cell lines. (a) Five human T-lymphocyte cell lines (CEM, CEMx174, A-3, D1.1, and E6-1) were transduced with a GFP-expressing HIV-1 vector at MOIs 0.2, 0.5, and 1.0. TRIM5α and CypA RNA levels at the time of transduction were evaluated by reverse transcription polymerase chain reaction, and TRIM5α protein levels were evaluated by Western blot analysis. The transduction efficiency (%GFP) was evaluated by flow cytometry 3 days after transduction. Additionally, cell proliferation was evaluated by fold expansion of cell counts during 3-day culture. (b–d) In all cell lines, %GFP was positively correlated with fold expansion of cell counts, while %GFP was negatively correlated with both TRIM5α RNA levels and TRIM5α protein levels. No correlation was observed between %GFP and CypA RNA levels.

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TRIM5α Effects for CD34+ Cell Transduction

relatively similar (1.65-fold magnitude). In all cell lines, %GFP was negatively correlated with both relative TRIM5α RNA levels (R2 = 0.83; P < 0.05 at MOI 1) and TRIM5α protein levels (R2 = 0.80; P < 0.05 at MOI 0.5), while %GFP was positively correlated with fold expansion of cell counts (R2 = 0.93; P < 0.01 at MOI 0.5; Figure 3b,c). No correlation was observed between %GFP and CypA RNA levels (R2 = 0.15; P = 0.52 at MOI 0.5; Figure 3d).

Human TRIM5α expression levels were negatively correlated with transduction efficiency in human CD34+ cells Human CD34+ cells from 14 individual donors were transduced with the HIV-1 vector at MOI 50 following 1 day prestimulation

a

(Figure 4a). TRIM5α RNA levels, TRIM5α protein levels, and CypA RNA levels were evaluated as before. A large variability of %GFP was observed among individual donors (8.5–35.8%). TRIM5α RNA levels were also widely variable among individual donors, while CypA RNA levels were relatively similar in human CD34+ cells (27.7-fold magnitude for TRIM5α, 6.27-fold magnitude for CypA). In donor cells, %GFP was negatively correlated with both TRIM5α RNA levels (R2 = 0.33; P < 0.05) and TRIM5α protein levels (R2 = 0.44; P < 0.01), while a positive correlation was observed between %GFP and fold expansion of cell counts (R2 = 0.33; P < 0.05), similar to the results from the T-lymphocyte cell lines (Figure 4b,c). Again, no correlation was observed between %GFP and CypA RNA levels (R2 = 0.02; P = 0.65; Figure 4d).

b

40

y = 12.2 + 8.40x R 2 = 0.330*

HIV-1 vector Human CD34+ cells

Medium change

%GFP

1 day

1 day Prestimulation

c

30

Transduction (MOI = 50) 2 days

20

RT-qPCR Western blot

Cell counts flow cytometry

10

TRIM5α and CypA levels

Fold expansion and %GFP

0

0

0.5

1.0

1.5

40

40

y = 24.4–0.628x R 2 = 0.332*

y = 40.8–15.7x R 2 = 0.473** 30 %GFP

%GFP

30

20

10

0

20

10

0

5

10

15

20

25

30

0

0

Relative TRIM5α RNA levels

d

2.0

Cell counts (fold expansion)

0.5

1.0

1.5

2.0

2.5

Relative TRIM5α protein levels

40

* P < 0.05, **P < 0.01

%GFP

30

20

10

0

0

1

2 3 4 Relative CypA RNA levels

5

Figure 4  Negative effects of TRIM5α levels on transduction in human CD34+ cells. (a) Human CD34+ cells from 14 individual donors were transduced with the HIV-1 vector at MOI 50 following 1 day prestimulation. The TRIM5α RNA levels, TRIM5α protein levels, and CypA RNA levels were evaluated at the time of transduction, and transduction efficiency (%GFP) and cell counts were evaluated 3 days after transduction. (b–d) In all donor cells, a positive correlation was seen between %GFP and fold expansion of cell counts (R2 = 0.33; P < 0.05), while %GFP was negatively correlated with both TRIM5α RNA levels (R2 = 0.33; P < 0.05) and TRIM5α protein levels (R2 = 0.47; P < 0.01). No correlation was observed between %GFP and CypA RNA levels (R2 = 0.02; P = 0.65).

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These data demonstrate that human TRIM5α expression levels negatively impact HIV-1 vector transduction efficiency in both human T-lymphocyte cell lines and CD34+ cells, while human CypA transcript levels do not impact transduction efficiency in either system.

Reduction of TRIM5α expression by short hairpin RNA (shRNA) increased transduction efficiency for a human T-lymphocyte cell line We developed stable E6-1 T-lymphocyte cell lines containing an expression cassette for human TRIM5α-specific shRNA

a

(shTRIM5α). The shTRIM5α-expressing cell lines (N = 10) and original E6-1 cells (N = 1) were transduced with a YFP-expressing HIV-1 vector at MOI 0.5. TRIM5α RNA levels, TRIM5α protein levels, and CypA RNA levels were evaluated before transduction, and transduction efficiency (%YFP) and cell counts were evaluated 3 days after transduction (Figure 5a). TRIM5α RNA levels varied widely among cell clones, while both CypA RNA levels and fold expansion of cell counts were relatively similar (10.0-fold magnitude for TRIM5α, 2.71-fold magnitude for CypA, 2.48-fold magnitude for cell counts). We observed a negative correlation between %YFP and TRIM5α protein levels (R2 = 0.44; P < 0.05),

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Figure 5  Positive effects of TRIM5α depletion on transduction for a human T-lymphocyte cell line. (a) We developed stable E6-1 T-lymphocyte cell lines containing an expression cassette for human TRIM5α-specific short hairpin RNA (shTRIM5α). The shTRIM5α expressing cells (N = 10) and original E6-1 cells (N = 1) were transduced with a yellow fluorescent protein (YFP)-expressing HIV-1 vector at MOI 0.5. The TRIM5α RNA levels, TRIM5α protein levels, and CypA RNA levels were evaluated before transduction, and transduction efficiency (%YFP) and cell counts were evaluated 3 days after transduction. (b–d) TRIM5α RNA levels had large difference among cell clones, while CypA expression levels and fold expansion of cell counts were relatively similar (10.0-fold magnitude for TRIM5α, 2.71-fold magnitude for CypA, 2.48-fold magnitude for cell counts). We observed a negative correlation between %YFP and TRIM5α protein levels (R2 = 0.44; P < 0.05), while there was a negative correlation between %YFP and TRIM5α RNA levels when excluding two outliers (R2 = 0.89; P < 0.01). The %YFP was not correlated with both CypA RNA levels (R2 = 0.24; P = 0.11) and fold expansion of cell counts (R2 = 0.24; P = 0.10).

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while a negative correlation was observed between %YFP and TRIM5α RNA levels when excluding two outliers (R2 = 0.89; P < 0.01; Figure 5c). %YFP was not correlated with either CypA RNA levels (R2 = 0.24; P = 0.11) or fold expansion of cell counts (R2 = 0.24; P = 0.10; Figure 5b,d). These data confirm that human TRIM5α expression levels affect HIV-1 vector transduction efficiency in human cells.

Higher TRIM5α transcript levels were observed in primitive human CD34+CD38− cells CD34+CD38− cells were sorted from CD34+ cells derived from three separate donors, and these cells were transduced with a GFP-expressing lentiviral vector following 1 day prestimulation. TRIM5α and CypA transcript levels were evaluated in both CD34+CD38− cells and bulk CD34+ cells at the time of transduction. We observed 38-fold higher TRIM5α transcript levels in CD34+CD38− cells (P < 0.05), compared to bulk CD34+ cells, while there was a small difference (threefold) of CypA transcript levels between both cells (P < 0.05; Figure 6a). The CD34+CD38− cells showed relatively lower %GFP, compared to bulk CD34+ cells (Figure 6b). These data suggest that more primitive CD34+CD38− cells have higher TRIM5α expression, which might result in lower transduction efficiency with lentiviral vectors.

DISCUSSION

We demonstrated that rhesus TRIM5α genotypes influence the variability of lentiviral vector transduction efficiency in transduced CD34+ cells and peripheral blood cells in transplanted rhesus macaques. In rhesus cell lines expressing TRIM5α Mamu-4, -5, and TrimCyp resulted in higher transduction efficiency with both χHIV and SIV vectors, while the HIV-1 vector had efficient transduction in only the TrimCyp-expressing cell line. This suggests that the animals with TRIM5α Mamu-4, -5, or TrimCyp might be more suitable when efficient transduction with χHIV and SIV vectors in hematopoietic cells is crucial to the aims of the experiments. However, rhesus macaques have two alleles of the TRIM5α gene, resulting in various genotypes (21 total genotypes). This leads to difficulty evaluating the variability of transduction efficiency in rhesus macaques. Additionally, %GFP in CD34+

a

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cells in vitro will likely be affected by not only TRIM5α variations but also other factors, such as mobilized CD34+ cell number or mobilization regimen. Therefore, we used multivariate analysis to demonstrate that TRIM5α type Mamu-4 significantly influences efficient transduction with the χHIV vector for granulocytes and lymphocytes in transplanted animals. The average data also suggest that Mamu-4 might result in the most efficient transduction with the χHIV vector for rhesus hematopoietic repopulating cells. Mobilized rhesus CD34+ cell number and mobilization regimen (G+SCF versus G+PL) also influenced transduction efficiency in rhesus CD34+ cells. Previously, we demonstrated that mobilized rhesus CD34+ cell numbers were significantly associated with efficient transduction with γ-retroviral vectors in hematopoietic repopulating cells of transplanted animals (unpublished results). We also previously reported that transplanted animals mobilized with G+SCF had higher lentiviral vector transduction efficiency in hematopoietic repopulating cells compared to animals mobilized with G+PL.15 Interestingly, when %GFP in vitro was included in multivariate analysis, mobilization regimen was no longer significant for %GFP in granulocytes and lymphocytes, while mobilized CD34+ cell number still significantly affected %GFP in granulocytes and lymphocytes. This suggests that the mobilization regimen might affect transduction efficiency in CD34+ cells, whereas mobilized CD34+ cell number might improve engraftment of transduced CD34+ cells in transplanted animals. Three transplanted animals lacked engraftment of genemodified cells (<1%) even with sufficient %GFP (36–86%) in the transduced CD34+ cells. Two (#15 and #16) of the three animals received low dose (4Gy) of total body irradiation instead of ablative dose (5Gyx2), which we recently determined to be insufficient to permit reliable engraftment of gene-modified CD34+ cells and induce immunological tolerance to GFP transgene.25 The TrimCyp-expressing cell line was efficiently transduced with the HIV-1 vector as well as the χHIV and SIV vectors. TrimCyp is a chimeric protein of TRIM5α and CypA, caused by a point mutation of the 3′ splicing signal in TRIM5α intron 6;16–18 this fusion cannot recognize the HIV-1 capsid for antiviral defense.28 This is consistent with transplantation data in pigtailed

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Figure 6 Higher TRIM5α transcript levels in human CD34+CD38− cells. (a) We evaluated CypA and TRIM5α RNA levels in three donors of CD34+CD38− cells and bulk CD34+ cells, and these cells were transduced with a GFP-expressing lentiviral vector. We observed 38-fold higher TRIM5α RNA levels in CD34+CD38− cells (P < 0.05), compared to bulk CD34+ cells, while there was a small difference (threefold) of CypA RNA levels between both cells (P < 0.05). (b) The CD34+CD38− cells had relatively lower transduction efficiency (%GFP), compared to bulk CD34+ cells. Standard deviations are shown as error bars.

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macaques (most animals have homozygous TrimCyp), in which HIV-1 vectors efficiently transduce hematopoietic cells.29 Although animals with homozygous TrimCyp can be efficiently transduced with HIV-1 vectors, this animal model cannot be used to investigate preclinical safety of lentiviral transduction under TRIM5α restriction (as humans do not express the TrimCyp allele). We evaluated the frequency of TRIM5α allele types in both National Institutes of Health Animal Center and Oregon National Primate Research Center. These rhesus colonies have a slightly higher frequency of type Q (Mamu-4 and -5) compared to another reported colony (New England Primate Research Center).20 However, all rhesus colonies had similarly high frequencies of TFP (Mamu-1, -2, and -3) and Q, compared to the frequency of TrimCyp.20,21 This suggests that a large variability of lentiviral transduction efficiency might be widely observed in rhesus macaques independent of transduction protocol or vector, and HIV-1 vectors will likely be unable to efficiently transduce hematopoietic cells in most rhesus macaques. Several human TRIM5α coding polymorphisms are known; however, unlike in rhesus macaques, the polymorphisms were thought to be insufficient to account for the variation in HIV-1 infectivity in human cells. Most of the known coding polymorphisms in human TRIM5α have little effects on HIV-1 infectivity.22,23 An H43Y polymorphism in the RING region of TRIM5α was reported to reveal an increase of HIV-1 infectivity but occurs at low frequency in the population (~5%).22,23 Additionally, an artificial mutation in R332P in the SPRY region was shown to increase HIV-1 infectivity; however, this polymorphism in human TRIM5α is not observed in nature.30,31 Therefore, we evaluated polymorphisms in the promoter region upstream of TRIM5α gene. We found three polymorphisms, and of these, two polymorphisms modestly contributed to higher TRIM5α protein levels and lower transduction efficiency (Supplementary Figure S1). Additionally, we evaluated known CypA polymorphisms in coding and promoter regions, and two polymorphisms in the promoter region were detected (C1604G and A1650G).26,27 However, these polymorphisms did not affect either %GFP or CypA RNA levels in human CD34+ cells (Supplementary Figure S2). These data suggest that human TRIM5α and CypA polymorphisms in the coding regions do not influence HIV-1 vector transduction efficiency in human CD34+ cells, and that TRIM5α expression levels might account for the transduction efficiency. We then evaluated TRIM5α transcript and protein levels in human T-lymphocyte cell lines and human CD34+ cells. We demonstrated a large difference of TRIM5α RNA levels among human T-lymphocyte cell lines and among 14 donors of human CD34+ cells, while little difference was seen in CypA RNA levels. TRIM5α RNA and protein expression levels were negatively correlated with transduction efficiency in both human T-lymphocyte cell lines and donor CD34+ cells, suggesting that TRIM5α expression levels influence the variability of HIV-1 vector transduction efficiency in human CD34+ cells. This hypothesis was confirmed experimentally reducing human TRIM5α expression levels using TRIM5α-specific shRNA. Additionally, fold expansion of cell counts was positively correlated with transduction efficiency for human CD34+ cells. Previously, we demonstrated that nuclear transport is a limiting 356

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step in lentiviral transduction for human CD34+ cells.32 Cell expansion media increases transduction efficiency in human CD34+ cells, but engraftment ability of human CD34+ cells was reduced, when evaluated by humanized xenograft mice.14 This suggests that faster cell cycle could increase transduction efficiency with lentiviral vectors in human CD34+ cells but could also decrease engraftment of CD34+ cells. We evaluated TRIM5α transcript levels and transduction efficiency in human CD34+CD38− cells, which contain more primitive hematopoietic cell population than bulk CD34+ cells. The CD34+CD38− cells had higher TRIM5α transcript levels and relatively lower transduction efficiency (Figure 6). These data are consistent with the rhesus transplantation data, which showed lower %GFP in peripheral blood cells in vivo, compared to CD34+ cells in vitro (Table 3). These data suggest that the lower transduction efficiency observed among primitive hematopoietic repopulating cells transduced with lentiviral vectors which might be caused by high TRIM5α expression levels. In summary, rhesus TRIM5α genotypes (especially type Mamu-4) play an important role in allowing efficient lentiviral transduction for rhesus CD34+ cells in vitro and in vivo and also contribute to the variability of gene marking in the rhesus HSC gene therapy model. Human TRIM5α expression levels were negatively correlated with transduction efficiency of an HIV-1based lentiviral vector in both human T-lymphocyte cell lines and CD34+ cells. Our findings are important for both understanding and mitigating the variability of transduction efficiency for human and rhesus CD34+ cells.

MATERIALS AND METHODS

Lentiviral vector preparation. The self-inactivating HIV-1- and SIV-based

lentiviral vectors were prepared as previously described.15,33 The GFP- or YFP-expressing HIV-1 vector was prepared by cotransfection of the four plasmids: HIV-1 Gag/Pol, HIV-1 Rev/Tat, vesicular stomatitis virus glycoprotein envelope, and HIV-1 vector plasmids, while the SIV vector was prepared by SIV Gag/Pol, SIV Rev/Tat, vesicular stomatitis virus glycoprotein envelope, and SIV vector plasmids. The capsid region in the HIV-1 Gag/ Pol plasmid was replaced with that of an SIV capsid to develop chimeric HIV-1 vectors (χHIV vectors) that efficiently transduced both human and rhesus hematopoietic cells, as previously described (Figure 1a).13,14,34 Both HIV-1 and SIV vector systems were kindly provided by Arthur Nienhuis (St Jude Children’s Research Hospital, Memphis, TN).35,36

Lentiviral transduction for stable cell lines expressing rhesus or human TRIM5α. The stable Crandell-Rees Feline Kidney cell lines expressing six

types of rhesus TRIM5α were kindly provided by Welkin Johnson (Harvard Medical School, Southborough, MA).20 Mock cells encoding neomycin resistance gene were utilized as a control. The rhesus TRIM5α-expressing cell lines were transduced with GFP-expressing HIV-1, χHIV, and SIV vectors at MOIs 0.5, 1, 2, and 5 in 12-well dishes including 1 × 10e5 cells in 1 ml Dulbecco’s modified Eagle medium with 10% fetal bovine serum and 8 µg/ml polybrene in triplicate (N = 3). Three days after transduction, transduction efficiency was evaluated by %GFP using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Five T-lymphocyte cell lines (CEM, CEMx174, A-3, D1.1, and E61) were obtained from the American type culture collection (Manassas, VA), and these cell lines were transduced with the GFP-expressing HIV-1 vector at MOIs 0.2, 0.5, and 1.0 in 12-well dishes including 1 × 10e5 cells in 1 ml Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum and 8 µg/ml polybrene in triplicate (N = 3; Figure 3a). www.moleculartherapy.org  vol. 22 no. 2 feb. 2014

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Three days later, %GFP was evaluated by flow cytometry, and cell counts were evaluated with trypan blue stain. Rhesus HSC transplantation with lentiviral transduction. We previ-

ously developed a large animal model for HSC transplantation with lentiviral transduction in rhesus macaques (Figure 2a).13–15 The G-CSF (Amgen, Thousand Oaks, CA) and SCF (kindly provided by BioVitrum AB, Stockholm, Sweden) or G-CSF and plerixafor (Genzyme, Cambridge, MA)-mobilized rhesus CD34+ cells were cultured in X-VIVO10 media (Lonza, Basel, Switzerland) containing SCF, FMS-like tyrosine kinase 3 ligand (FLT3L), and thrombopoietin (TPO; all 100 ng/ml; R&D Systems, Minneapolis, MN). After 1 day prestimulation, the CD34+ cells were transduced with GFP− (or YFP−) expressing χHIV vectors at MOI 50, and the next day, these cells were infused into the rhesus macaque following two consecutive days of 5Gy total body irradiation (5Gyx2) for a total dose of 10Gy. A small amount of the transduced CD34+ cells were cultured in vitro in fresh media with the same cytokines. Two to 3 days later, transduction efficiency in transduced CD34+ cells was evaluated for %GFP (or YFP) by flow cytometry. Six months after transplantation, %GFP was evaluated in granulocytes and lymphocytes of the transplanted animals. For the statistical analysis, animals transplanted in competitive repopulation assays were scored by doubling the %GFP derived from the half of CD34+ cells transduced with the χHIV vector. For in vivo data analysis, we excluded three animals that had no engraftment of gene-modified cells. Human CD34+ cell culture with lentiviral transduction. Human CD34+

cells were enriched from peripheral blood stem cells mobilized by G-CSF under a protocol approved by the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Disease and were stored in a −180 °C freezer. Frozen human CD34+ cells were quickly thawed in a 37 °C water bath and cultured on fibronectin (RetroNectin; Takara, Shiga, Japan) coated plates using serum free X-VIVO10 media (Lonza) containing SCF, FLT3L, and TPO (all 100 ng/ml, R&D Systems) in triplicate (N = 3; Figure 4a).15 After 1 day prestimulation, these cells were transduced with the GFP-expressing HIV-1 vector at MOI 50. The TRIM5α RNA levels, TRIM5α protein levels, and CypA RNA levels were evaluated at the time of transduction, and %GFP and cell counts were evaluated 3 days after transduction. Additionally, human CD34+ cells were stained by phycoerythrinconjugated anti-CD34 antibody (clone 563; BD Biosciences) and allophycocyanin-conjugated anti-CD38 antibody (clone T10; Miltenyi Biotec, Auburn, CA), and the CD34+CD38− fractions were sorted by FACSAria II flow cytometer (BD Biosciences).

DNA sequencing of rhesus TRIM5α, human TRIM5α, and human CypA.

Rhesus TRIM5α genotyping was performed as previously described.16,20 Briefly, genomic DNA was extracted from peripheral blood cells, and the C-terminal SPRY region of TRIM5α was amplified and sequenced using the primers (forward: 5′-CAG TGC TGA CTC CTT TGC TTG-3′ and reverse: 5′-GCT TCC CTG ATG TGA TAC-3′). The amino acid sequences (numbers 307, 327, 333, and 339) determined TRIM5α types Mamu-1or -2, -3, -4, and -5 (Table 1). TrimCyp is defined by a G-to-T substitution in the 3′ splice acceptor in intron 6 of rhesus TRIM5α. We sequenced the promoter region (~1k bases) upstream of human TRIM5α gene (forward primer: 5′-GAG GCT ATG GTA CAT GGA CCA CAG TTC AGC3′, reverse primer: 5′-AGG AAA TTC TTG CTC ACA CTC AGG GCA GGA-3′).37 We sequenced known human CypA polymorphisms in the promoter region (forward primer: 5′-CTT CCG TCT ATA GGC CAG ATG C-3′, reverse primer: 5′-CAC TTT CTG GGC CCC ATT CC-3′), exon 4 (forward primer: 5′-ATT CCA GGG TTT ATG TGT CAG G-3′, reverse primer: 5′-CCA GCT AAG GGC CAA GTT TC-3′), and exon 5 (forward primer: 5′-GCT ACC TTT CTC GTC TTG GTT C-3′, reverse primer: 5′-CCA CAG TCA GCA ATG GTG ATC TTC-3′).26,27

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Reverse transcription real-time polymerase chain reaction. Total RNA

was extracted from human T-lymphocyte cell lines and human CD34+ cells at the time of transduction. The RNA was converted into complementary DNA using random hexamers and reverse transcriptase (SuperScript III First-Strand Synthesis System; Life Technologies, Grand Island, NY). The cDNA was then used as a template, and specific sequences were amplified by real-time polymerase chain reaction (Mx3000P QPCR Systems; Agilent Technologies, Santa Clara, CA) using TRIM5α-specific probe/primers and CypA-specific probe/primers.38 TaqMan Ribosomal RNA control reagents (Applied Biosystems, Foster City, CA) were used for standardization. Western blot analysis. Human T-lymphocyte cell lines and human CD34+

cells were washed once in phosphate-buffered saline and lysed in RIPA buffer (Sigma Aldrich, St Loius, MO). Total cell lysates were electrophoresed on NUPAGE 4–12% Bis (2-hydroxyethyl)-Tris (tris-hydroxymethyl-aminomethane) gels (Life Technologies), and then transferred to polyvinylidene difluoride membranes (Life Technologies). The membranes were blocked in tris-buffered saline with 0.1% Tween-20 and 5% blotting grade blocker nonfat dry milk (Bio-Rad, Hercules, CA) for 1 hour, and then incubated with anti-TRIM5α (1:1,000) antibody (clone 3B11H2; Abcam, Cambridge, MA) at 4 °C overnight. After three washes, blots were incubated with horseradish peroxidase–conjugated antimouse antibody (Roche Applied Science, Indianapolis, IN), and then developed using a SuperSignal West Pico Chemiluminescent detection system (Thermo Scientific, Rockford, IL). Glyceraldehyde 3-phosphate dehydrogenase expression levels served as an internal control using anti-glyceraldehyde 3-phosphate dehydrogenase antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Western blot signals were quantitated using Quantity One software (Bio-Rad).

Lentiviral transduction to produce stable E6-1 cell lines with reduced human TRIM5α expression. We developed stable E6-1

T-lymphocyte cell lines containing an expression cassette for human TRIM5α-targeting shRNA, which was designed based on an shRNA against rhesus TRIM5α with one base mutation to match the human TRIM5α sequence (modified shRNA 3: 5′-GCC TTA CGA ACT CTG AAA C-3′).11 The relative TRIM5α expression levels were evaluated by reverse transcription polymerase chain reaction and Western blot analysis before transduction, and these cells were transduced with a YFP-expressing HIV-1 vector at MOI 0.5 in 12-well dishes including 1 × 10e5 cells in 1 ml Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum and 8 µg/ml polybrene in triplicate (N = 3; Figure 5a). Two days after transduction, transduction efficiency was evaluated by %YFP in flow cytometry, and cell counts were evaluated with trypan blue stain. Statistical analysis. Statistical analyses were performed using the JMP

9 software (SAS Institute, Cary, NC). The averages in various conditions were evaluated by Dunnett’s test (one-way analysis of variance for one control). Two averages were evaluated by the Student’s t-test. The multivariate analysis was performed using stepwise regression. Correlations were evaluated by R2 in linear regression and P values in correlation coefficient. A P value of <0.01 or 0.05 was deemed significant. Standard errors of the mean are shown as error bars in all figures.

SUPPLEMENTARY MATERIAL Figure S1. TRIM5α polymorphisms in the promoter region modestly affect the protein levels and lentiviral transduction in human CD34+ cells. Figure S2.  CypA polymorphisms in the promoter region have little effect on transduction in human CD34+ cells. Figure S3. Large variability in transduction efficiency among T-lymphocyte cell lines.

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ACKNOWLEDGMENTS This work was supported by the intramural research program of the National Heart, Lung, and Blood Institute and the National Institute of Diabetes, Digestive, and Kidney Diseases at the National Institutes of Health. We thank Mark E. Metzger, Allen E. Krouse, Aylin C. Bonifacino, Barrington E. Thompson, and Sandra D. Price for the animal care and handling of the animal samples. We thank Kayo Uchida for her assistance in the statistical analysis. We thank J. Philip McCoy and Heidi Sardon for cell sorting. The authors declare no competing financial interests.

References

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