Application of SFHR to Gene Therapy of Monogenic Disorders

Application of SFHR to gene therapy of monogenic disorders

KK Goncz1, NL Prokopishyn2, BL Chow2, BR Davis2 and DC Gruenert1 1Human Molecular Genetics Unit, Department of Medicine, University of Vermont, Burlington, VT, USA; and 2Gene-Cell, Inc., Houston, TX, USA

Gene therapy treatment of disease will be greatly facilitated to gene targeting. The potential for one gene targeting tech- by the identiﬁcation of genetic mutations through the Human nique, small fragment homologous replacement (SFHR) to Genome Project. The speciﬁc treatment will ultimately the gene therapy treatment of sickle cell disease (SCD) is depend on the type of mutation as different genetic lesions presented. Successful conversion of the wt-␤-globin locus to will require different gene therapies. For example, large a SCD genotype of human lymphocytes (K562) and rearrangements and translocations may call for comp- progenitor/stem hematopoietic cells (CD34+ and lin-CD38Ϫ) lementation with vectors containing the cDNA for the wild- was achieved by electroporation or microinjection small DNA type (wt) gene. On the other hand, smaller lesions, such as fragments (SDF). the reversion, addition or deletion of only a few base pairs, Gene Therapy (2002) 9, 691–694. DOI: 10.1038/sj/gt/3301743 on single genes, or monogenic disorders, lend themselves

Keywords: SFHR gene therapy of monogenic disorders; gene targeting; sickle cell disease; ␤ (beta) globin

Introduction ability to use SFHR will ultimately depend on the efficiency of delivering DNA fragments to the diseased Monogenic, autosomal recessive disorders are good can- organs. For both MD and CF, conventional wisdom sug- didates for gene therapy because a normal phenotype can gests that DNA fragments will need to be delivered sys- be restored in diseased cells with only a single normal temically or in situ to reach the diseased organs. The suc- copy of the mutant gene. One potential gene therapy cess of this type of delivery is currently dependent on technique is small fragment homologous replacement the development of DNA carrier vehicles that can deliver (SFHR). In this technique, the endogenous genetic DNA to specific organs in situ. For monogenic hematopo- mutation responsible for the disorder is corrected by ietic disorders, such as sickle cell anemia and thalasse- small fragments of DNA (~400–800 bp) that are intro- mia, the diseased organ, the blood system, is continually duced into cells. The fragments are essentially homolo- regenerated from blood stem cells in the bone marrow. gous to the sequence of the mutant gene except that they Since the bone marrow can be removed from the patient, code for the normal, rather than the mutant sequence. To DNA fragments can be delivered, or transfected, into date, SFHR has been successfully applied in model sys- cells ex vivo. The treated cells can then be returned to the tems by correcting engineered mutations in the Zeocin patient in an autologous transplantation. As such, it is 1 2 resistance gene, the green fluorescent protein and the possible to capitalize on DNA delivery techniques that luciferase gene (M Bennett, personal communication). In have already been optimized for in vitro transfection. To addition, SFHR has been used to target and successfully determine if SFHR can be used as a gene therapy option modify specific sequences of endogenous genomic loci for ex vivo treatment of sickle cell anemia, SFHR- that are the source of several monogenic disorders using mediated modification of the endogenous ␤-globin gene in vitro and/or in vivo models. Specifically, the dystro- locus was analyzed in normal human lymphoblasts phin gene in the mdx mouse model of Duchenne muscu- (K562), as well as in hematopoietic progenitor/stem cells 3 lar dystrophy (MD) mouse, the cystic fibrosis transmem- (CD38Ϫ/linϪ or CD34+). The DNA fragments (559 bp) brane conductance regulator (CFTR) gene in cystic used for transfection in these experiments are exactly 4–6 7 Tב fibrosis (CF) human epithelial cells, and normal mice, homologous to normal ␤-globin (␤A) except for an A ␤ as well as the -globin gene in human hematopoietic conversion located in the sixth codon of the gene (ie sic- 8 progenitor/stem cells. kle or ␤S genotype) and a silent mutation that introduces While SFHR has shown potential as a genetic therapy a unique AflII site. The cells were transfected with DNA technique for treatment of monogenic disorders, the fragments using either electroporation or glass-needle mediated microinjection. Electroporation of K562 cells with the 559 bp small Correspondence: KK Goncz, Human Molecular Genetics Unit, Depart- DNA fragment resulted in the conversion of endogenous A S ment of Medicine, University of Vermont, HSRF 222, 149 Beaumont Ave, ␤ globin to ␤ globin (Figure 1). The conversion was suc- Burlington VT 05405, USA cessful when cells were electroporated with DNA at a Application of SFHR to gene therapy of monogenic disorders KK Goncz et al 692

Figure 2 Expression of ␤S globin mRNA after transfection of K562 cells with small DNA fragments. PCR amplification was performed on mRNA- derived cDNA (SuperScript II, Invitrogen, La Jolla, CA, USA) using either the allele-specific sense primer (SC9S) or the non-allele specific sense primer (SC5) (5’-acatttgcttctgacacaactgtg-3’) and the anti-sense primer SC14 (5’-actggtggggtgaattctttgc-3’). (a) Analysis of mRNA-derived cDNA from K562 cells transfected with 2 million fragments. Lane 1, allele-specific (SC9A/SC14) PCR amplification from transfected cells shows the expected 382 bp band; lane 2, sample from lane 1 without Ϫ Figure 1 SFHR-mediated modification of endogenous genomic ␤-globin reverse transcription ( RT); lane 3, non-allele-specific PCR amplification sequences in K562 cells as determined by allele-specific amplification of from transfected cells showing the expected 430-bp band; lane 4, sample isolated DNA. The fragments used for these experiments were prepared from lane 3 –RT; lane 5, non-allele-specific PCR amplification of non- Ϫ by PCR amplification based on previously published methods.5,23 Briefly, transfected control cells; and lane 6, sample from lane 5 RT. (b) Com- ␤ preparative amounts of the 559-bp fragment were generated with the sense parison of -globin expression of transfected K562 and non-transfected ␤ primer SC11 (5’-aaagtcagggcagagccatcta-3’) and the anti-sense primer control normal cells (A181 -MEL). Non-allele-specific PCR amplification ␤ SC12 (5’-gggaaagaaaacatcaagggtc-3’) using, as a template, a cloning vec- of mRNA-derived cDNA from K562 cells (lane 1), A181 -MEL cells (lane tor that contains a sequenced copy of the 559-bp fragment. The PCR con- 2) and K562 DNA (lane 3). ditions were initial denaturization, 95°C/2 min followed by 25 cycles of denaturization, 95°C/30 s, annealing, 65°C/30 s, extension, 72°C/1 min with a 7 min extension in the final cycle. The PCR mixture contained 200 ␮M dNTPs, 200 nM each primer, 1 × Taq Polymerase Buffer (Perkin

Elmer, Applied Biosystems, Foster City, CA, USA), 2 mM MgCl2 and 0.5 units of Taq (Perkin Elmer) in a 100 ␮l reaction. The resulting DNA fragments were purified by ammonium acetate and ethanol precipitation and resuspended in water. K562 cells (ATCC CCL-243) were transfected with denatured fragment by electroporation. Briefly, 8 × 106 cells and fragment were resuspended in 800 ␮l in 0.4 cm electroporation chambers and placed on ice. The cells were then electroporated using a BTX 300 Gene Pulser at a setting of 500 ␮F and 240 V for 200 ms. After electroporation, the cells were placed on ice for 10 min and then resuspended in DMEM for normal growth. DNA and RNA was isolated from the cells 1 week after transfection5 and subjected to PCR amplification. For these reactions, the sense primer is allele-specific for either ␤A globin (SC9A) ␤S (5’-accatggtgcacctgactcctca-3’)or globin (SC9S) (5’- Figure 3 SFHR-mediated modification of endogenous genomic ␤-globin accatggtgcacctgactcctct-3’) and the anti-sense primer (SC4) (5’-aac- sequences in hematopoietic progenitor/stem cells. Human hematopoietic gatcctgagacttccacact-3’) is non-allele specific. Lane 1, DNA from K562 progenitor/stem cells (CD34+ or CD38Ϫ/linϪ) were isolated from cord cells transfected with 1 million fragments per cell; lane 2, DNA from blood as previously described.24 The progenitor/stem cells were plated on K562 cells transfected with 2 million fragments per cell; lane 3, control to retronectin-coated dishes into a cloning ring. The cells were then K562 DNA; lane 4, control normal DNA; lane 5, control sickle DNA; microinjected with a glass pulled capillary tube loaded with ~5 ␮l of DNA ␤A and lane 6, water. Samples in the top row were amplified with specific fragment (~1 ␮g/␮l) and Oregon Green Dextran (OGD) suspended in ␤S primers (593 bp) and in the bottom (593 bp). PBS. Successful microinjection and cell viability was determined by OGD fluorescence. After microinjection, the cells were transferred in bulk to 48- well plates and grown in either maturation media (IMDM, BSA/Insulin/transferrin, LDL, SCF, Flt-3, GCSF, GMCSF, IL-6, IL-3) × 6 concentration of either 1 or 2 10 fragments per cell. or in Methocult assays until analysis. PCR amplification was performed The genomic conversion resulted in expression of sickle on 20 ␮l of cell lysate that was created by resuspending 105 cells in 50 ␤-globin RNA (Figure 2a; lane 1). The results indicate that ␮l of lysis buffer containing 500 ␮g/ml proteinase K, 0.45% Nonidet P- expression of the ␤-globin gene locus is increased in 40, 0.45% Tween 20 and 5 mM Tris (pH 8.3) for 60 min at 56°C followed ° Ϫ Ϫ transfected versus non-transfected K562 cells (Figure 2a; by 10 min at 95 C. Lane 1, control CD38 /lin cells; lane 2, bulk sample 1; lane 3, bulk sample 2; lane 4, bulk sample 3; lane 5, control normal lane 3 versus lane 5). Normally, K562 cells express little ␤ DNA; lane 6, control sickle DNA. Samples on the top were amplified with if any -globin, although there are K562 subclones that ␤A specific primers (593 bp) and ␤S on the bottom (593 bp). do express ␤-globin9 and endogenous ␤-globin expression can be induced in K562 cells by transcription factors.10 A non-specific band (~550 bp) that is present cannot, therefore, be attributed to contaminating DNA after amplification of mRNA-derived cDNA from trans- (Figure 2b; lane 3). fected K562 cells has not yet been identified. The band Microinjection of hematopoietic progenitor/stem cells appears to be larger by the same amount as both the with the 559 bp small DNA fragment also resulted in suc- allele-specific sickle ␤-globin amplicon (Figure 2a; lane 1) cessful conversion of endogenous ␤A globin to ␤S globin and the non-allele specific ␤-globin amplicon (Figure 2a; (Figure 3). Successful conversion (lane 3) is shown along lane 3). The band does not appear after amplification of with unsuccessful conversion (lanes 2 and 4) of cells that mRNA-derived cDNA from non-transfected K562 (Figure were also injected with ~1000 copies/fl (0.2–2.5 fl injected 2a; lane 5) or A181␤-MEL cells (Figure 2b; lane 2) and per cell). It is possible that other factors such as cell type

Gene Therapy Application of SFHR to gene therapy of monogenic disorders KK Goncz et al 693 Table 1 Microinjection conditions for hematopoietic progenitor/stem cells

Sample Cellsplated Cells injected Successful injections Viable injections Time in culture(medium)a Final cell count

1 CD38ϪlinϪ 1000 88 59 ~35 69 days (MM) 1.5 × 106 2 CD34+ 1000 172 107 60 34 days (Meth) 2.7 ×106 3 CD38ϪlinϪ 300 256 194 49 33 days (Meth) 3.5 ×106 a MM, Maturation medium; Meth, Methocult assay. or the fraction of viable cells can explain the variability poietic cell gene therapy include the retrovirus,14,15 lenti- in conversion (Table 1). Conversion of the silent mutation virus,16,17 foamy virus18 and adeno-associated virus.19,20 was not detected by AflII restriction digestion of allele- There have however, been a number of issues that have specific PCR amplicons in the experiments presented arisen with these viral vector systems that have under- here (data not shown). This result indicates that conver- mined their effectiveness. These limitations include the sion of specific genomic base pairs by SFHR is not linked. lack of long-term expression, position effects on gene In this study, we have shown that the endogenous ␤- expression and gene silencing due to the site of viral inte- globin locus can be modified by SFHR in clinically rel- gration and/or the presence of viral promoter/enhancers evant cells. Specifically, we have shown that the driving cell expression. The primary advantage is that endogenous ␤A globin allele of both normal lymphoblast transfection efficiency can approach 100%. and CD34+ and/or CD38ϪlinϪ cells can be converted to For SFHR to be a potential gene therapy, the number a ␤S globin allele. In addition, the genetic conversion of SFHR-modified cells needs to approach therapeutic leads to the expression of ␤S globin mRNA. The percent levels. It has been estimated that only ~40 000 number of converted alleles in all of the successful experi- CD38Ϫ/linϪ/CD34+/KDR+ bone marrow cells would ments was in the range of 1 to 10% based on the sensi- be required to insure reconstitution of a child’s blood sys- tivity of PCR amplification.11 Similar efficiencies have tem.21,22 If these cells were successfully microinjected been observed when the ␤A globin allele was converted with small DNA fragments, the patient could be pro- to ␤S in CD34+-enriched cells using chimeric RNA/DNA vided with therapeutically beneficial levels of gene- hybrid oligonucleotides.12 However, the number of oli- repaired cells. Given current technology of microinjection gonucleotides per cell that were used in those experi- systems and future improvements, the likelihood of ments was approximately 100 times greater than the SFHR as a gene therapy technique for sickle cell anemia number of fragments per cell in the K562 experiments and other hematopoietic diseases is highly probable. and approximately 6 × 104 times greater than the number per cell in the hematopoietic progenitor/stem cell References experiments. While it is difficult to accurately quantify the percent 1 Colosimo A et al. Targeted correction of a defective selectable conversion of alleles in these experiments, the results in marker gene in human epithelial cells by small DNA fragments. Figure 1 suggest that SFHR-mediated conversion is dose- Mol Ther 2001; 3: 178–185. dependent. K562 cells exposed to 2 million fragments per 2 Thorpe P, Stevenson B, Gohil A, Porteous D. Towards CFTR cell showed a stronger conversion than those exposed to gene correction: a comparison of two key strategies. Ped Pulmon 2000; (Suppl. 20) 241. 1 million fragments per cell. Again, these data correlate 3 Kapsa R et al. In vivo and in vitro correction of the mdx dystro- with previous work that shows dose-dependent conver- phin gene nonsense mutation by short-fragment homologous 12 sion by RNA/DNA hybrid oligonucleotides. In this replacement. Hum Gene Ther 2001; 12: 629–642. study, percent conversion increased linearly up to 90 4 Goncz K, Kunzelmann K, Xu Z, Gruenert D. Targeted replace- million RNA/DNA oligos per cell reaching a plateau ment of normal and mutant CFTR sequences in human airway through to 300 million oligos per CD34+-enriched cell. In epithelial cells using DNA fragments. Hum Mol Genet 1998; 7: our studies, it is impossible to determine the actual num- 1913–1919. ber of fragments or oligos delivered per cell in the K562 5 Kunzelmann K et al. Gene targeting of CFTR DNA in CF epi- or CD34+ experiments because not all DNA fragments are thelial cells. Gene Therapy 1996; 3: 859–867. 6 Sangiuolo F et al. In vitro correction of CF cells using SFHR tech- delivered to the nuclei of electroporated cells.13 The fact nique. Pediatric Pulmonary 2000; (Suppl. 20) 240. that we observed correction after microinjection of 7 Goncz K et al. Expression of DeltaF508 CFTR in normal mouse fragment directly into the nucleus hematopoietic lung after site-specific modification of CFTR sequences by progenitor/stem cells indicates that SFHR-mediated SFHR. Gene Therapy 2001; 8: 961–965. modification can occur with as little as 2500 fragments 8 Goncz K. Conversion of normal ␤-globin to sickle ␤-globin by (Figure 3). Moreover, the modification is permanent as small fragment homologous replacement. Blood 2000; 96: 379b. these cells went through at least 10 doublings by the time 9 Mookerjee B, Arcasoy M, Atweh G. Spontaneous delta- to beta- they were isolated (Table 1). At the time of cell harvest, globin switching in K562 human leukemia cells. Blood 1992; 79: there was less than the equivalent of 10 fragments per 820–825. cell; thus spurious amplification from contaminating 10 Mahajan M, Weissman S. DNA-dependent adenosine triphos- 4 phatase (helicase-like transcription factor) activates beta-globin fragment cannot account for results. transcription in K562 cells. Blood 2002; 99: 348–356. Currently, gene therapy of hemoglobinopathies has 11 Goncz K, Gruenert D. Progress toward nucleotide sequence ␤A been focused on the introduction of globin gene modification at the genome level: small fragment homologous sequences ex vivo via virus-based expression vectors. The replacement. Biotechnology 2001; 3: 113–120. viral vector systems that have been proposed for hemato- 12 Xiang Y et al. Targeted gene conversion in a mammalian CD34+

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