WO2008136670A2

WO2008136670A2 - Improved methods and means for lentiviral gene delivery

Info

Publication number: WO2008136670A2
Application number: PCT/NL2008/050269
Authority: WO
Inventors: Nico Peter Van Til; Monique Maria Andrea Verstegen; Gerard Wagemaker
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2007-05-02
Filing date: 2008-05-02
Publication date: 2008-11-13
Also published as: WO2008136670A3

Abstract

The invention relates to the field of gene therapy and more in specific to lentiviral gene delivery vehicles and methods for efficient transduction of lentiviral gene delivery vehicles into hematopoietic stem cells and their descendants. Preferably, the invention provides in one of its embodiments a method of gene transfer into e.g. pluripotent hematopoietic stem cells and their descendants, enabling successful transduction of cells, including transplantable cell populations comprising hematopoietic stem cells that give rise to progeny expressing the transduced gene(s). The invention further comprises a method for treating a variety of hereditary and acquired human disease by transfer of therapeutically active genes into hematopoietic stem cells. As a non-limiting example, the invention shows that symptoms associated with Pompe disease are (completely) reduced and/or alleviated by treatment of a subject suffering from Pompe disease withJiematopoietic stem cell transduced with an alpha- glucosidase comprising lentiviral vector.

Description

Title: Improved methods and means for lentiviral gene delivery

The invention relates to the field of gene therapy and more in specific to lentiviral gene delivery vehicles and methods for efficient transduction of lentiviral gene delivery vehicles into hematopoietic stem cells and their descendants. Preferably, the invention provides in one of its embodiments a method of gene transfer into e.g. pluripotent hematopoietic stem cells and their descendants, enabling successful transduction of cells, including transplantable cell populations comprising hematopoietic stem cells that give rise to progeny expressing the transduced gene(s). The invention further comprises a method for treating a variety of hereditary and acquired human disease by transfer of a therapeutically active gene into hematopoietic stem cells. As a non-limiting example, the invention shows that symptoms associated with Pompe disease are (completely) reduced and/or alleviated by treatment of a subject suffering from Pompe disease with hematopoietic stem cell transduced with an alpha-glucosidase comprising lentiviral vector.

Successful correction of inherited hematopoietic disorders by gamma retrovirus vector-mediated ex υiυo hematopoietic stem cell (HSC) gene- modification has been demonstrated for X-linked severe combined immunodeficiency SCID and adenosine deaminase deficiency (ADA-SCID). However, efficacy also revealed potential genotoxicity, essentially due to insertional transactivation of neighbouring genes by retroviral enhancer elements, resulting at a very low frequency in aberrant expression of neighboring genes and thereby in clonal dominance/leukemogenesis. Vector systems with a reduced risk profile are therefore essential in the further development of gene therapy for inherited diseases. HIV-I derived self- inactivating lentiviral vectors (LV) display a reduced risk pattern due to deletion of enhancer regions and a more favourable integration pattern as compared to gamma-retroviruses. With current LV transduction protocols, a high proportion of gene-modified HSCs requires a high multiplicity of infection (MOI), or rather a high concentration of transducing units (TU), which results in an undesirable high number of transgene copies per cell.

The aim of the current invention is to improve lentiviral vector transduction efficiency of HSC and preferably to limit the number of integrations per cell. To test optimal transduction conditions the inventors used VSV-G packaged LV expressing EGFP. The inventors observed that retronectin and growth factor supported overnight transduction of lineage depleted (lin ^Λ) mouse bone marrow cells (BMC) with low cell-density, but a high MOI of 30 yielded approximately 60% EGFP⁺ cells.

Unexpectedly, at an MOI of 1, the transduction efficiency of lirr⁷- cells could be markedly improved from 7% to 54% by increasing the cell density from 2 x 10⁴/ml to 6 x 10⁵/ml with a proportional increase of TU/ml.

The increased cell density/viral load did not enhance the mean fluorescence of EGFP⁺ cells, suggesting that integrations per cell did not increase. Q-PCR confirmed an average of approximately 1 provirus copy per cell.

Mouse lin '- BMCs transduced with LV at MOI 2 resulted in 98% EGFP⁺ cells, with sustained reconstitution of up to 80% EGFP⁺ blood cells after testing the gene-modified cells in a competitive repopulation assay in vivo.

Similar results were obtained for human CD34⁺ umbilical cord blood (UCB) cells. Transduction efficiencies of up to 70% at 6 x 10⁵ cells/ml were observed in UCB cells by increasing TU/ml with proportionaHncreases in cell density, maintaining a low MOI of 1.

In summary, the inventors of the current invention show that an increase in cell density of target cells with a fixed ratio of transducing units (TU) per cell (or MOI) improved vector transduction efficiency of HSC. In a first embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle.

Surprisingly, this controlled transduction method results in the presence of essentially one therapeutic gene integration per target cell, which has been impossible with other lentiviral transduction methods. Moreover, another desirable effect of the method according to the invention is that only limited amount of therapeutic gene vector batches need to be produced, thus saving considerable time in respect of quality control and production time.

As used herein, the term transduction refers to the stable transfer of genetic material from a viral particle to a hematopoietic stem cell genome. The hematopoietic system produces perpetually large numbers of blood cells, which have a limited life span and need to be constantly renewed throughout the life of a mammal. This renewal is maintained through proliferation and differentiation of a small number of hematopoietic stem cells in the bone marrow. The definition of stem cells is not always clear within the art. Herein a functional definition is used, which defines hematopoietic stem cells as those cells capable of (long term) reconstitution of a hematopoietic system. This definition is often felt to include at least some early progenitor cells. Since blood cells virtually reach every organ, hematopoietic stem cells are a highly suitable target for gene therapy for a variety of hereditary and acquired diseases within and outside the hematopoietic system. Unfortunately, until the present invention, lentiviral mediated gene transfer has met with only limited success due to the difficulty of obtaining sufficient numbers of successfully transduced, transplantable, single-copy of therapeutic gene containing, long-term repopulating hematopoietic stem cells.

In more detail, HSC are stem cells and the early precursor cells which give rise to all the blood cell types that include both the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets and some dendritic cells) and lymphoid lineages (T-cells, B-cells, NK-cells, some dendritic cells). The definition of HSC has undergone considerable revision in the last two decades. The hematopoietic tissue have cells with long term and short term regeneration capacities and committed multipotent, oligopotent and unipotent progenitors. HSC can be obtained from different sources and are, for example, found in the bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Cells can be obtained directly by removal from the hip using a needle and syringe, or from the blood following pre-treatment with cytokines, such as G-CSF (granulocyte colony stimulating factor), that induces cells to be released from the bone marrow compartment into the blood (mobilized peripheral blood). Other sources for clinical and scientific use include umbilical cord blood, and placenta. For experimental purposes, fetal liver, fetal spleen and AGM (Aorta-gonad-mesonephros) of animals are also useful sources of HSCs.

HSC are phenotypically identified by their small size, lack of lineage (lin) markers, low staining (side population) with vital dyes such, as rhodamine 123 (rhodamine^DULL, also called rho¹⁰) or Hoechst 33342, and presence of various antigenic markers on their surface many of which belongs to the cluster of differentiation series, such as: CD34, CD38, CD90, CD133, CD105, CD45 and also c-kit- the receptor for stem cell factor. The_hematopoietic stem cells are negative for the markers which are used for detection of lineage commitment and are thus called Lin-, and during their purification by FACS, a bunch of up to 13 to 14 different mature blood-lineage marker e.g. CD13 and CD33 for myeloid, CD71 for erythroid, CD 19 for B cells, CD61 for megakaryocyte etc for humans; and, B220 (murine CD45) for B cells, Mac-1 (CDllb/CD18) for monocytes, Gr-I for Granulocytes, Terll9 for erythroid cells, 117Ra, CD3, CD4, CD5, CD8 for T cells etc for mice) antibodies are used as a mixture to deplete the lin+ cells or late multipotent progenitors (MPP)s. The VSV-g pseudotyped lentiviral vector described in the experimental part can in principle transduce all kinds of cell types because the VSV-g provides the lentiviral particles with a broad tropism. In a preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said HSC are murine, human or primate cells. More preferred said HSC are bone marrow cells, umbilical cord blood cells or mobilized peripheral blood stem cells. Even more preferred said HSC are CD34 positive (CD34⁺) cells or CD34 positive / CD38 dull cells.

Gene delivery vehicles of lentiviral origin are all vehicles comprising genetic material and/or proteinaceous material derived from lentiviruses.

Typically the most important features of such vehicles are the integration of their genetic material into the genome of a target cell and their capability to transduce stem cells. These elements are deemed essential in a functional manner, meaning that the sequences need not be identical to lentiviral sequences as long as the essential functions are present. The methods of the invention are however especially suitable for recombinant lentiviral particles, which have most if not all of the replication and reproduction features of a lentivirus, typically in combination with a producer cell having some complementing elements. Normally the lentiviral particles making up the gene delivery vehicle are replication defective on their own.

It is clear to the skilled person that a gene delivery vehicle is intended to read on any vehicle capable of delivering genetic material to a target cell, whether the genetic material is actually a gene, an antisense molecule or a cosuppressive nucleic acid (encoding molecule), etc. Useful nucleic acids to be provided to target cells, e.g. stem cells are well known in the art and include such molecules as to replace inborn errors/deficiencies of the hematopoietic system, which may include hemoglobin genes and their regulatory elements for the thalassemia's and sickle cell anemia's and sequences to repair the various forms of severe combined immunodeficiency, such as caused by adenosine deaminase deficiency and that known as severe X linked immunodeficiency, or genes encoding enzymes for diseases known as lysosomal storage diseases, such as Hurler's, Hunter's, Krabbe's and in particular Gaucher's disease and metachromatic leukodystrophy, or by introducing sequences that confer resistance of the progeny of hematopoietic stem cells to infectious agents, such as HIV, as well as the introduction of suicide genes for cancer therapy and marker genes to track the progeny of transplanted normal and/or malignant hematopoietic stem cells.

The prior art in respect of lentiviral transduction. of hematopoietic stem cells typically use a target cell concentration of for example IxIO⁵ to 4xlO⁵ in combination with a relatively high multiplicity of infection (i.e., around 20 to 100). Another published used ratio of target cell concentration versus MOI is 5-2OxIO⁴ cells versus a MOI of 100.

It is clear from the experimental part herein that an increase of 2xlO⁴ to 6xlO⁵ cells/mL at an MOI of 1 resulted in an increase of transduction percentage..

In a preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said amount of HSC is at least 6xlO⁵/mL. This concentration of cells is especially useful for human and murine cells. For rhesus macaque cells, transduction percentages comparable to the mentioned human and murine percentage were obtained by using a cell density of 2.5χlO⁶ cells/mL. It is clear for the skilled person that a suitable target cell density depends on the kind of host used as a source, for example human, murine or primate.

The terms "amount of HSC" or "number of HSC" or "concentration of HSC" or "density of HSC" are used herein interchangeably. The upper limit of the concentration of target cells is for example dependent on the amount of cells present in an organism used for isolation. For clinical purposes this will be approximately IxIO⁸ cells. Based on the herein disclosed results an upper limit of 10⁷ to 2xlO⁷ should be feasible. In a preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said amount of HSC is maximal 10⁷ to 2xlO⁷ cells/mL. In yet another preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said amount of HSC is at least 6xlO⁵ cells/mL and maximal 10⁷ to 2xlO⁷ cells/mL.

The herein used concentration of target cells (i.e. HSC) are for example obtained by isolating said target cells from a suitable source (for example bone marrow or umbilical cord or peripheral blood) and subsequently centrifuging the obtained cells to a pellet and finally resuspending the obtained pellet in a small(er) volume (when compared to the original, i.e., before centrifugation, volume).

The term multiplicity of infection (MOI) or concentration of transducing units (TU) are used interchangeable herein and refers to the number of viruses or virus particles that infect a single cell on average. In yet another preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said relatively low amount of transducing units per cell is 1-10.

It is clear to the skilled person that an MOI of between 0.1 and 0.9 can equally well be used. However, in such a case, a method of the invention preferably further comprises selecting the cells that have been transduced. Preferably, the MOI of the used viral particles is determined on

HeLa cells.

In a preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said relatively low amount of transducing units per cell is 1-5, more preferably 1-4, even more preferably 1-3 and most preferably 1-2.

Methods and means (such as producer cells) for producing the desired lentiviral particles are well known in the art. Examples of preferred producer cells are 293T cells that are cα-transfected with VSV-G (Vesicular stomatitis virus- G-protein envelope). Other pseudotypes used in this setting are RDl 14 (Feline-immunodeficiency virus).

In a most preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said amount of HSC is at least 6xl0⁵ cells/mL and wherein said relatively low amount of transducing units per cell is 1-10. Typically, the term "gene delivery vehicle of lentiviral origin" refers to a gene delivery of any lentiviral origin. Currently, five serogroups of lentiviruses are recognized, reflecting the vertebrate hosts with which they are associated (primates, sheep and goats, horses, cats, and cattle): bovine, equine, feline, ovinecaprine and HIV serogroup. In a preferred embodiment, the used "gene delivery vehicle of lentiviral origin" is a "gene delivery vehicle of HIV lentiviral origin" and even more preferred are HIV-I derived self-inactivating lentiviral vectors.

In yet another preferred embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, which is an ex vivo or in vitro method.

The invention also includes compositions obtainable by the methods of the invention. Thus included are compositions comprising HSC transduced with a gene delivery vehicle of lentiviral origin.

The invention further provides a composition comprising lentiviral particles wherein said composition has a relatively low amount of transducing units per amount of target cell. Preferable, said lentiviral particles are gene delivery vehicles and capable of transducing HSC and/or progenitor cells. Even more preferably said lentiviral particles are capable of transducing bone marrow cells, umbilical cord blood cells or mobilized peripheral blood stem cells.

The term multiplicity of infection (MOI) or concentration of transducing units (TU) are used interchangeable herein and refers to the number of viruses or virus particles that infect a single cell on average. In yet another preferred embodiment, the invention provides a composition comprising lentiviral particles wherein said composition has a relatively low amount of transducing units per amount of target cell, wherein said relatively low amount of transducing units per cell is 1-10. Preferably, the MOI of the used viral particles is determined on

HeLa cells.

In a preferred embodiment, the invention provides a composition comprising lentiviral particles wherein said composition has a relatively low amount of transducing units per amount of target cell, wherein said relatively low amount of transducing units per cell is 1-5, more preferably 1-4, even more preferably 1-3 and most preferably 1-2.

The invention also provides the pharmaceutical use of the described compositions, particularly in the treatment of diseases having a genetic component, such as the various genetic hemoglobin orders, the large group of rare diseases collectively known as severe combined immune deficiencies, the group of lysosomal storage diseases, especially with a strong hematopoietic and/or visceral expression, such as Gaucher' s disease, but also possibly Hurler's or Pompe's diseases, as well as in the treatment of infectious disease, notably HIV infection, or cancer. Typically the use of a composition comprising lentiviral particles involves the transduction of HSC such as bone marrow cells, umbilical cord blood cells or mobilized peripheral blood stem cells (for example CD34 positive target cells). Such transduced cells are typically made ex vivo and are also part of the present invention. Thus the invention provides a composition for the treatment of a hereditary disease or a pathological condition related to a genetic defect or a genetic aberration, comprising a plurality of, for example, CD34 positive cells transduced with a composition of lentiviral particles according to the invention, or a composition for the treatment of a hereditary disease or a pathological condition related to a genetic defect or a genetic aberration, comprising a plurality of, for example, CD34 positive cells, said composition being obtainable by a method according to the invention.

In yet another preferred embodiment, the invention provides the use of a composition as described above in the preparation of a medicament for the treatment of a hereditary disease or pathological condition related to a genetic defect or a genetic aberration.

Such a use is particularly useful in the treatment of diseases having a genetic component, such as the various genetic hemoglobin orders, the large group of rare diseases collectively known as severe combined immune deficiencies, the group of lysosomal storage diseases, especially with a strong hematopoietic and/or visceral expression, such as Gaucher's disease, but also possibly Hurler's or Pompe's diseases, as well as in the treatment of infectious disease, notably HIV infection, or cancer. Typically the use of a composition comprising retroviral particles involves the transduction of, for example, CD34 positive target cells. Such transduced cells are typically made ex vivo and are also part of the present invention.

To provide insight in the useful applications of the current invention the following paragraphs are directed to genetic diseases in general and to the applicability of the current invention in respect of lysosomal storage diseases. Treatment directed to alleviation of symptoms associated-with Pompe's disease is provided as a non-limiting example.

Rare diseases, of which 80% are genetic diseases, affect some 6% of the human population, amounting in Europe to around 30 million people. Since many result in chronic disability and cost-intensive care, the impact is disproportional and may well be over 20% of health care costs. Most of these diseases lack a curative intervention, are managed by symptomatic treatment, and are amenable to gene therapy with curative intent. At the state-of-the-art, gene therapy is within reach for a variety of monogenic inherited rare diseases, clinical implementation requiring selection of diseases in which (1) the genetic defect is identified, (2) the diagnosis is made sufficiently early for meaningful therapeutic intervention, (3) a specific animal model is available for efficacy and safety evaluation, (4) strict regulation of transgene product levels is not required, (5) the transgene produces levels sufficient for sustained alleviation of symptoms or cure, (6) adverse immune responses to the transgene product are either not expected or do not interfere with efficacy. Lysosomal storage diseases, a group of over 50 different enzyme deficiencies, meet these requirements. The deficiencies result in intracellular deposition of storage material and loss of cell function. Clinical features display a broad spectrum of severity reflecting the extent of the enzyme deficiency. For some, allogeneic hematopoietic stem cell (HSC) transplantation (alloSCT) is an effective treatment, even in diseases with brain involvement. However, alloSCT requires intensive conditioning, an HLA identical donor, is dependent on endogenous enzyme levels of the hematopoietic system which may be insufficient, carries immunological risks with undue morbidity and mortality, and is applicable only to selected patients and diseases. The alternative enzyme replacement (ERT) therapy is currently or shortly available for some ten of the lysosomal enzyme deficiencies, however, requires life-long administration, does not provide cure, is not effective in all patients, does not pass the blood brain barrier, and is extremely expensive. Ectopic production of enzyme by autologous HSC is anticipated to combine the advantages of SCT with those of enzyme therapy into a single curative procedure at limited cost. Pompe's disease (glycogen storage disease type II; acid maltase deficiency) is a lethal autosomal recessive disorder caused by acid a- glucosidase deficiency, which results in generalized storage of glycogen in many tissues such as brain, liver, spleen, kidney, endothelial cells, and most prominently in skeletal muscles, heart and smooth muscle. Symptoms arise from muscle weakness and wasting. Infants with complete enzyme deficiency present shortly after birth and become completely quadriplegic within 8 months. The natural course of Pompe's disease is invalidating by progressive loss of mobility and respiratory function and is lethal for severely affected infants in the first year of life. Older children and adults with residual activity show a more protracted course of disease and become wheelchair bound, dependent on artificial ventilation and die from cardiac/respiratory failure ranging from early childhood to late adulthood. Recently, the EMEA and FDA have approved ERT with recombinant human alpha-glucosidase for all patients. Enzyme therapy is well tolerated, its beneficial effects are unquestionable and prolong life significantly, but do not guarantee long-term symptom free survival. The therapy prolongs life of severely affected infants significantly and improves motor outcome, but during 8 years of experience approximately half of the treated infants ultimately died and all develop some form of residual disease. The beneficial effect of enzyme therapy in older children and adults still needs to be validated. The development of alternative gene therapy therefore would significantly contribute to the survival of 50% of the severely affected infants, and eventually would be particularly beneficial as a single treatment option for the juvenile form of Pompe's disease.

Previous experiments in which adenovirus and adeno-associated virus based vectors were used for systemic delivery of acid a-glucosidase gene constructs in mice have demonstrated its efficacy in long lasting reduction of glycogen storage. Importantly, its expression in muscle was not essential. Also, the liver could function as an enzyme -secreting depot supplying corrective enzyme to muscle and other tissues, warranting exploration of liver directed systemic administration of therapeutic vector. However, these vectors are applied via an in vivo approach and as a result shedding problems may arise as well as adverse immune reactions. Moreover, permanent integration is not necessarily obtained and hence treatment must be repeated in time.

In the experimental part herein, we demonstrate in an available mouse model that high level expression of human acid a-glucosidase in descendents of HSC transduced with SIN-lentiviral vectors provides a valid treatment option for alleviation of clinical symptoms of Pompe's disease. This approach does not require the administration of virus particles to the patient (compared to ade no- based treatment). The invention therefore provides the use of a composition as described above in the preparation of a medicament for the treatment of a lysosomal storage disease or a pathological condition related to a lysosomal storage disease. In a preferred embodiment, said lysosomal storage disease is Pompe disease, i.e. in a preferred embodiment, the invention provides the use of a composition as described above in the preparation of a medicament for the treatment of Pompe disease.

A typical pathological condition associated with Pompe disease is an increased storage of glycogen, which causes progressive muscle weakness throughout the body. As a result thereof-cardiac as well as respiratory complications arise.

The particulars of the present invention are further used to develop a kit, preferably a closed clinical grade device, which comprises means to purify and then transduce target cells (hematopoietic stem / progenitor cells). Thus, the invention further comprises a kit comprising means to isolate and/or purify hematopoietic stem cells and a therapeutic gene delivery vehicle.

For selection purposes special columns are already on the market (Miltenyi; for research purposes Mini-Midi and-Maxi columns, for the clinical setting CliniMacs columns). However, other means for isolating and/or purifying HSC are equally suitable for incorporation in a kit.

The other component of the described kit is a gene delivery vehicle that comprises a therapeutically interesting gene. Preferably, said gene delivery vehicle is of lentiviral origin. Typically the most important features of such vehicles are the integration of their genetic material into the genome of a target cell and their capability to transduce stem cells. These elements are deemed essential in a functional manner, meaning that the sequences need not be identical to lentiviral sequences as long as the essential functions are present. The methods of the invention are however especially suitable for recombinant lentiviral particles, which have most if not all of the replication and reproduction features of a lentivirus, typically in combination with a producer cell having some complementing elements. Normally the lentiviral particles making up the gene delivery vehicle are replication defective on their own. It is clear to the skilled person that a gene delivery vehicle is intended to read on any vehicle capable of delivering genetic material to a target cell, whether the genetic material is actually a gene, an antisense molecule or a cosuppressive nucleic acid (encoding molecule), etc. Useful nucleic acids to be provided to target cells, e.g. stem cells are well known in the art and include such molecules as to replace inborn errors/deficiencies of the hematopoietic system, which may include hemoglobin genes and their regulatory elements for the thalassemia's and sickle cell anemia's and sequences to repair the various forms of severe combined immunodeficiency, such as caused by adenosine deaminase deficiency and that known as severe X linked immunodeficiency, or genes encoding enzymes for diseases known as lysosomal storage diseases, such as. Hurler's, Hunter's, Krabbe's and in particular Gaucher's disease and metachromatic-leukodystrophy, or by introducing sequences that confer resistance of the progeny of hematopoietic stem cells to infectious agents, such as HIV, as well as the introduction of suicide genes for cancer therapy and marker genes to track the progeny of transplanted normal and/or malignant hematopoietic stem cells.

In a preferred embodiment, the invention comprises a kit comprising means to isolate and/or purify hematopoietic stem cells and a therapeutic gene delivery vehicle, further comprising instructions to transduce an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle. Moreover, the instructions can provide guidelines in respect of suggested reaction/transduction/culture conditions such as temperature, medium and incubation time.

The currently used lentiviral transduction systems are based on essentially random integration of the to be transferred nucleic acid sequences into the genomic sequence of the target cells (hematopoietic stem / progenitor cells). However the transduction method of the invention is not limited to this, because the method of the invention can equally well be performed in a transduction method in which the transferred nucleic acid sequence is incorporated into the genome of the target cell via homologous recombination processes.

Moreover, two general approaches can be used to correct defective genes within stem cells: gene addition and genome editing. Gene addition involves the delivery of corrective DNA (usually composed of the entire coding region of a gene and appropriate regulatory sequences) that compensates for or overrides the defective gene. The defective gene, unless it is completely absent, remains in the affected cells. In contrast, genome editing uses DNA repair and/or homologous recombination processes to correct an existing defective gene sequence so that the defective or mutated area of the gene is restored to a corrected normal state. Repairing the defective sequence itself maintains the corrected genetic material within its normal chromatin environment, ensuring appropriate genetic regulation and expression in the cell. Genome editing may be the only suitable strategy in situations in which mutant gene product exercises a dominant negative influence over the normal gene product.

Hence, in yet another embodiment, the invention provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle, wherein said gene delivery vehicle is provided with means for homologous recombination. Examples of such means are nucleic acid sequences encoding (at least part of) a gene involved in a hereditary disease, such as X-linked SCID, sickle cell anemia, and lysosomal enzyme deficiencies. The underlying mutation(s) in such hereditary disease is/are known or can easily be determined on a case-by-case basis.

Editing of the human genome in viυo is somewhat hindered by the low frequency of homologous recombination. This can be circumvented by using specially engineered 'zinc-finger' nucleases as molecular scissors to cut DNA inside cells at a specific sequence. The DNA break is then patched using new genetic information. The efficiency of this process combined with the ability to design zinc-finger nucleases that target almost any DNA sequence mean that genome editing in human cells is likely to become- an important research tool and potentially a powerful way of treating disease.

In yet another embodiment, the features of the present invention, i.e. efficiency of transduction is greatly enhanced by increasing the target cell density at low multiplicity of infection, are also useful in genome editing. The invention thus provides a method for transducing hematopoietic stem cells (HSC) with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells with a relatively- low amount of transducing units per cell of said gene delivery vehicle, wherein said gene delivery vehicle is provided with means for genome editing. An example of such a means is a Zinc finger endonuclease.

The invention will be explained in more detail in the following, non- limiting examples. Experimental part Materials and methods

Production of lentiυiral vectors. Third generation lentiviral vector batches were produced by standard transient calcium phosphate transfection of HEK 293T cells as described before ¹^⁵, subsequently ultracentrifugated and titers were determined by end- point titration on HeLa cells. We used the packaging plasmids pMDL-g/pRRE, pMD2-VSVg and pRSV-REV in combination with the self inactivating lentiviral transfer vectors pRRL.PPT.SF.EGFP.WPRE4*.SIN (SF-GFP), containing the HIV central polypurine tract, the spleen focus forming virus promoter or the phosphoglycerate kinase (PGK) promoter driving EGFP expression (PGK-GFP). In addition, the lentiviral vector contained a modified woodchuck posttranslational regulatory element (WPRE4*) ⁴ containing 4 deleted ATG-sites and a large deletion of the Woodchuck hepatitis X-protein sequence.

Enrichment of stem cells.

Mouse Un-/ - bone marrow cells Lineage depleted (lin-/-) mouse bone marrow cells were obtained with the mouse hematopoietic progenitor (stem) cell enrichment set according to the manufacturer's protocol (BD Biosciences).

Murine lin-/- were cultured in our standard stem cell medium (Stem cell activating; SAF) ³ (see below) supplemented with 100 ng/ml murine stem cell factor (SCF), 10 ng/ml murine thro mbopoie tin (TPO) and 50 ng/ml human recombinant fms-like tyrosine kinase 3-ligand (Flt3L). Rhesus monkey bone marrow cells

Low-density cells were isolated using Ficoll separation (1.077 g/cm², Nycomed, Pharma, AS, Oslo, Norway) and cryopreserved until subset purification or directly continued towards purification and transduction.

Subset purification

Purification of CD34⁺ rhesus cells was performed by positive selection using Dynalbeads (Dynal, Oslo, Norway). Briefly, low-density cells were incubated with an IgG2A antibody against CD 34 (mAb 561; from G. Gaudernack and T. Egeland, Rikshospitalet, Oslo, Norway) covalently linked to rat anti-mouse IgG2A beads. CD34⁺ cells devoid of the CD34-antibody were recovered using polyclonal antibodies against the Fab part of the CD34 antibody (Detachebead, Dynal). Purified rhesus CD34+ cells were analyzed by flow cytometry. Rhesus CD34+ cells were cultured in stem cell medium, supplemented with human Flt3-L (50 ng/ml, Thousand Oaks, CA, USA), rhTPO (10 ng/ml, Genentech, South San Francisco, CA, USA) and SCF (100 ng/ml).

Human umbilical cord blood cells

UCB samples were obtained after informed consent in conformity with legal regulations in The Netherlands from placentas of full-term normal pregnancies. Mononucleate d cells were isolated by Ficoll density gradient centrifugation (1.077 g/cm², Nycomed .Pharma AS, Oslo, Norway), and were cryopreserved in 10% dime thy lsulphoxide, 20% heat-inactivated fetal calf serum (FCS) and 70% Hanks Balanced Salt Solution (HBSS, Gibco, Breda, The Netherlands) at -196°C. After thawing by stepwise dilution in HBSS containing 2% FCS, the cells were washed with HBSS containing 1% FCS.

Subset purification

CD34⁺ cells were purified according to the manufacturer's instructions by magnetic selection using a biotinylated CD34⁺ antibody (clone 12.8 CellPro Inc, Bothell, USA), streptavidin MicroBeads, and MAC separation columns (Miltenyi Biotec Inc, Auburn, USA). The percentage CD34⁺ cells in the unseparated population (unfractionated UCB) and in the purified CD34⁺ and CD34- fractions was determined by FACS-analysis with fluorescein isothiocyanate (FITC) conjugated antibodies against human CD34 (Becton Dickinson) for 30', on ice in HBN (HBBS, 2% (wt/vol) FCS, 0.05% (wt/vol) sodium-azide) containing 2% (vol/vol) normal human serum (NHS). The cells were washed twice and analysed by flow cytometry. The CD34⁺ cells were cultured in our standardized serum free medium (SAF, stem cell activating, see below) supplemented with human recombinant growth factors fetal liver tyrosine kinase 3-ligand (Flt3-L; 50 ng/ml, Amgen, Thousand Oaks, CA, USA), thrombopoietin (TPO; 10 ng/ml, R&D Systems, Abingdon, UK) and stem cell factor (SCF; 100 ng/ml, R&D Systems, Abingdon, UK).

Standard stem cell medium:

Dulbecco's modified Eagle's medium (Gibco, Life Technologies Inc., Paisley, Scotland) supplemented with 2.8xlO ⁴ M L-alanine, 3.3xlO ⁴ M L-asparagine, 2.3xlO ⁴ M L-aspartic acid,.5.8xlθ-⁴ M L-cysteine, 5-lxlO ⁴ M L-glutamic acid, 3.5 xlO ⁴ M L-proline, l.δxlO"⁵ M cholesterol, 4 μM cytidine, 4 μM adenosine, 4 μM uridine, 3.5 μM guanosine, 4.4 μM 2'-deoxycytidine, 4 μM

2'deoxyadenosine, 4 μM thymidine, 3.7 μM 2'deoxy guanosine, 1.2xlO ⁷ M d- - biotin, 1% fraction V BSA, (all Sigma-Aldrich, Zwijndrecht, The Netherlands), 1.SxIO-⁸ M vitamin BΪ2, 10 ³ M sodium pyruvate, 1.9xlO ² M glucose (Merck), 4.4xlO-² M NaHCO₃, 10-* M β-mercapto-ethanol, KF M Na₂SeO₃, 1.5xl0 ⁵ M linoleic acid (Merck, Darmstadt, Germany), 0.1 g/1 penicillin (Yamanouchi, Leiderdorp, The Netherlands), 10⁵ IE/1 streptomycin (Fisiopharma, Milano, Italy), 2xlO'⁶ M iron saturated transferrin (Serologicals Proteins Inc. Kankakee, IL, USA), at an osmolarity of 300 mOsm/1. Transduction of hematopoietic stem cells.

Transduction of the cells was started at the same day of enrichment of the stem-cells. For the in vitro experiments lentiviral vector supernatant was mixed with the enriched hematopoietic stem cells at increasing cell-density of 2xlO⁴, 6xlO⁴, 1.2xlO⁵, 2χl0⁵ or 6xlO⁵ in 1 ml culture medium and these cells were subsequently seeded in retronectin (human recombinant fibronectin fragment CH-296, Takara Bio Inc) coated 24-well culture plates (surface area 2cm²). The multiplicity of infection (the number of viral transducing units per cell) was maintained at 1. The EGFP positive fraction of the transduced cells was determined after at least 4 days after the start of transduction. In addition, rhesus CD34+ cells were also transduced in 96-well plates (surface area 0.32 cm²) at Ix 10⁵ and 5χlO⁵ cells in 200μL SAF medium.

In vivo efficacy of transplanted lentiviral vector transduced hematopoietic stem cell.

Lin-/- bone marrow cells were isolated as described above and transduced overnight at an MOI of 2. Subsequently, BALB/c mice were 6Gy irradiated and 3χlO⁶ or Ix 10⁶ cells were injected intravenously in the tail vein. Blood was collected by orbital puncture 4 times until 190 days after transplantation and EGFP expression was determined in leucocytes.

Materials and methods for the treatment of Pompe disease

Development of lentiviral vector plasmids

The α-glucosidase (GAA) cDNA was removed from the pSHAG2-GAA vector previously described⁷ and cloned in the pSUPER vector containing a polylinker with EcoRI and Xbal sites (pSli). The pSli-GAA plasmid was digested with Agel and Xbal. The GAA cDNA fragment was ligated into the Agel-Nhel sites of the pRRL.PPT.PGK.EGFP.WPRE4*.SIN lentiviral transfer vector (LV-PGK- GAA) after removal of the enhanced Green Fluorescent Protein (eGFP) cDNA. The Agel-Swal fragment from the LV-PGK-GAA was cloned in place of the EGFP of the pRRL.PPT.SF.EGFP.WPRE4*.SIN lentiviral transfer vector to result in LV-SF-GAA lentiviral vector. Lentiviral vector batches were produced as described above, except that the lentiviral vector preparations were titrated on mouse GAA '- mesenchymal stem cells.

Animals

All mice were of FVB background and GAA '- mice were described previously⁸. GAA ^Λ mice have a complete deficiency in the alpha- glucosidase gene and therefore mimic the early onset-type of the disease, in clinical symptoms, and in pathological as well as biochemical findings⁸. The animal experiments were approved by an ethical committee of Erasmus Medical Center, Rotterdam in accordance with legal regulations in The Netherlands.

Lentiviral hematopoietic stem cell transduction

Male GAA-'- mice were used as bone marrow donor for transplantation. Total bone marrow was flushed from both femora and tibias and of 8-12 week-old male GAA-/- mice, and lineage depleted (lirr'-) according to the manufacturer's protocol (BD). After enrichment, cells were transduced by LV-SF-GFP of LV- SF-GAA lentiviral vectors overnight at IxIO⁶ cells/mL at multiplicity of infection (MOI) of 9-10, in serum-free modified Dulbecco's (Stem Cell Activating, SAF) medium supplemented as described and containing growth factors (m-SCF lOOng/ml, hu-Flt3 50ng/ml, m-TPO lOng/ml). The following day, 6 Gy irradiated 8-12 week-old GAA '- mice were injected in tail vein with 5xlO⁵ transduced lin '- cells.

Enzymatic assays

All tissues were sonicated on ice until tissues were completely lysed (medium level, amplitude 5). Activity of GAAwas determined by fluorometry, according to a previous described protocol based on 4-methylumbelliferyl α-D- glucopyranoside (4MU, Sigma)⁹.

Glycogen content was determined as described ¹⁰ by treatment of samples by amyloglucosidase. Resulting glucose was measured by treatment of glucose- oxidase and 2,2'-azino-di-(ethyl-benzthiazolinsulfonate) (ABTS). Final glycogen content values were determined after untreated glucose levels were subtracted. Both GAA and glycogen assays were measured by VarioSkan. GAA activity and glycogen content were corrected for protein content using the bicinchoninic acid (BCA) protein assay (Pierce).

Measurement of cardiac hypertrophy

GAA^+/+, and GAA '- mice, LV-SF-GFP of LV-SF-GAA treated were sacrificed and both left ventricular and right ventricular mass were determined by echography.

Experimental part Results

In vitro lentiviral vector transduction efficiency of hematopoietic stem cells.

Mouse lin-/- cells could be efficiently transduced with SF-GFP and PGK-GFP lentiviral vectors. An increase of 2χlO⁴ to 6χlO⁵ Hn-/- cells/mL at an MOI of 1 resulted in an increase of 4% to 41% and 7% to 54% of EGFP positive cells for SF-GFP and PGK-GFP respectively (Figure 1). Similar transduction efficiencies could be achieved with UCB cells at 6χlO⁵ cells/mL, which were on average 62% for SF-GFP lentiviral vector transduction. In contrast, rhesus CD34+ cells transduced less efficiently, but on average 22% EGFP positive rhesus CD34+ cells were measured at an MOI of 1 at 6χlO⁵ cells/mL in 24 well plates (Figure 1). Higher cell-densities in 96 well plates (5xlO⁵ and 2.5χlO⁶/mL) at an MOI of 1 resulted 31 and 50% EGFP positive cells respectively (Figure 2). Lentiviral transduction efficiency in Retronectin-coated plates was not improved compared to non-coated plates. Increase of the MOI to 37 at 6xlO⁵ cells/mL markedly increased the transduction efficiency from 21% to 80%.

In vivo lentiviral vector transduction efficiency of hematopoietic stem cells.

Mouse lin-/- BMCs transduced with lentiviral vectors at an MOI of 2 resulted in 98% EGFP positive cells, with up to 80% EGFP positive leucocytes in peripheral blood 46 days after transplantation in irradiated mice (Figure 3). A large proportion of EGFP positive cells was also measured in erythrocytes and thrombocytes. Long-term EGFP positive cells were measured in leucocytes for up to 190 days after transplantation (Figure 4), which shows the potential of the high cell-density transduction efficiency at low MOI. Quantitative PCR oflentiviral integrations

We analysed the number of integrations in the transduced mouse lin-/- cells, rhesus macaque CD34⁺ cells and human CD34⁺ UCBs, transduced at an MOI of 1. The number of integrations was at maximum 3.1 integrations per cell at 6xlO⁵ cells/mL for mouse lin-/- cells. At a cell-density of 6xlO⁵ cells/mL in rhesus macaque cells the average number of integrations per cell was 1.1 and in human UCBs 2.7. All the lower cell-densities resulted in less integrations per cell compared to the highest cell-density (6xlO⁵ cells/mL).

Results treatment ofPompe disease

The stem cell gene therapy approach for Pompe's disease has been tested in experiments using the GAA-/- mouse model for Pompe's disease. Long-term (1 year) GAA activity in leukocytes was detected in ex vivo LV-SF-GAA-corrected mice (96.8±53 nmol 4MU/h/mg, n=31) compared to expression in LV-SF-GFP controls (1.4±0.8 nmol 4MU/h/mg, n=14, pO.OOl) (Figure 5), resulting in high levels of enzyme in affected organs (Figure 6). PAS staining demonstrated a near complete clearance of glycogen in liver, spleen and cardiac muscle (Figure 7). Also skeletal muscle displayed a significant reduction of glycogen, although not as prominent as the other tissues (Figure 7). The large activity increase of GAA in the heart tissue resulted in a near normalization of heart geometry and function as visualized by echography. Heart rate (beats per minute) was reduced in LV-SF-GFP mice at 6.5 months of age (315±11) and significantly improved in LV-SF-GAA mice (385+27), similar to heart rate in healthy mice 363+17. In addition, LV-SF-GFP mice develop ventricular hypertrophy due to cardiac muscle weakness. At 8 months after treatment, both the healthy mice and LV-SF-GAA mice had significantly reduced left ventricular weight (82.219.37 mg and 96.01±7.73 mg, respectively) compared to LV-SF-GFP mice (119.96+11.16 mg) (Figure 8). LV-SF-GAA treated mice older than 150 days showed significantly improved muscle strength determined by grip strength measurements. However, consistent with the glycogen clearance levels, healthy mice were significantly stronger than LV-SF-GAA treated mice. Further locomotor and respiratory functional tests are in progress. Adverse effects on the hematopoietic system were not observed.

We conclude that ex vivo hematopoietic system mediated gene therapy corrects defects in the Pompe mouse model, indicating that the approach provides a valid alternative for enzyme replacement therapy in the treatment of Pompe's disease. Codon optimization of the transgene is anticipated to provide a further elevation of enzyme levels.

Description of figures

Figure 1.

Increase in transduction efficiency of enriched hematopoietic bone marrow cells by increasing the cell-density (2χlO⁴ →6χl0⁵/mL in 24-well format) with a fixed MOI of 1. Increase in transduction efficiency is depicted for mouse lin -/- cells, rhesus CD34+ cells and CD34+ umbilical cord blood cells. These cells were transduced with lentiviral vectors containing the SF-EGFP or PGK- EGFP promoter cassettes.

Figure 2.

Increase in lentiviral vector transduction efficiencies of rhesus CD34+ cells at high cell-densities at a fixed MOI of 1. Retronectin is not required for efficient transduction of rhesus CD34+ cells. Filled bars: retronectin, open bars: no retronectin.

Figure 3.

Transplantation of transduced mouse lin-/- BMCs (MOI 2) in irradiated mice and the EGFP positive fraction in peripheral blood. A,B) Percentage of EGFP positive cells of cultured mouse lin-/- BMCs 6 days after transduction of a lentiviral vector containing the SF-EGFP promoter construct, C-H) Percentage of EGLFP positive cells in peripheral blood of mice 46 days after transplantation of transduced lin-/- BMCs: C₁D) Erythrocytes. E,F) Thrombocytes. G,H)^~ Leucocytes. A,C,E,G) Non-transduced controls.

Figure 4.

Long-term EGFP expression in mouse leucocytes after high cell-density ex viυo lentiviral vector transduction. Transplantation of transduced mouse lin-/- BMCs (MOI 2) in 6Gy irradiated mice and the EGFP positive fraction in leucocytes. Percentage of EGFP positive leucocytes in mice transplanted with 3xlO⁶ and Ix 10⁶ transduced lin-/- cells containing the SF-EGFP promoter construct. Long-term EGFP expression was observed until 190 days after transplantation.

Figure 5.

Long-term expression of GAA in blood. Long-term GAA activity was detected by enzymatic assay in leukocytes of GAA-/- LV-SF-GAA-treated mice (n=32) compared to LV-SF-GFP (n=15) treated mice. GAA activity of leukocytes in GAA+/+ mice (n=6) was comparable to GAA-/- mice.

Figure 6.

GAA activity in tissues. Upper graph. At 8 months after treatment high GAA activity was observed in leukocytes of GAA-/- LV-SF-GAA treated mice, as well as bone marrow (BM) and spleen. GAA+/+ mice GAA activity was detectable in BM and spleen, but this was low. No GAA activity was detected in GAA-/- LV- SF-GFP treated mice. Lower graph. High GAA activity was observed in GAA-/- LV-SF-GAA mice in heart, lung, diafragm, liver, stomach , uterus and quadriceps femoris (QF) similar to GAA+/+ mice.

Figure 7.

Glycogen content in affected tisues. Glycogen content of GAA-/- LV-SF-GAA treated mice was decreased in heart, lung, diafragm, liver, stomach and uterus and moderately reduced in quadriceps femoris (QF).

Figure 8.

Reduction in left ventricular cardiac hypertrophy after LV-SF-GAA treatment. Cardiac mass was determined in 10-month-old mice, eight months after hematopoietic transplantation. Left ventricular mass was significantly increased in GAA-/- LV-SF-GFP mice compared to GAA+/+ mice (*, p=0.001). In addition, GAA-/- LV-SF-GAA treated mice showed a significant reduction in left ventricular mass compared to GAA-/- LV-SF-GFP mice (f, p<0.05).

References

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Claims

1. A method for transducing hematopoietic stem cells with a gene delivery vehicle of lentiviral origin comprising contacting an increased amount of hematopoietic stem cells (HSC) with a relatively low amount of transducing units per cell of said gene delivery vehicle.

2. A method according to claim 1, wherein said HSC comprise bone marrow cells, umbilical cord blood cells or mobilized peripheral blood stem cells.

3. A method according to claim 1 or 2 wherein said HSC are CD 34 positive cells, or CD34 positive / CD38 dull cells.

4. A method according to any one of claims 1 to 3, wherein said amount of HSC is at least 6x105 cells/mL.

5. A method according to any one of claims 1 to 4, wherein said relatively low amount of transducing units per cell is 1-10.

6. A method according to any one of claims 1 to 5, wherein said gene delivery vehicle of lentiviral origin is a gene delivery vehicle of HIV lentiviral origin.

7. A method according to any one of claims 1 to 6 which is an ex vivo method.

8. A composition comprising lentiviral particles wherein said composition has a relatively low amount of transducing units per amount of target cell.

9. A composition according to claim 8, wherein said lentiviral particles are gene delivery vehicles.

10. A composition according to claim 8 or 9 for use as a pharmaceutical.

11. Use of a composition according to claim 8 or 9 in the transduction of hematopoietic stem cells.

12. A composition for the treatment of a hereditary disease or pathological condition related to a genetic defect or a genetic aberration, comprising a plurality of hematopoietic stem cells transduced with a composition according to claim 8 or 9.

13. A composition for the treatment of a hereditary disease or pathological condition related to a genetic defect or a genetic aberration, comprising a plurality of hematopoietic stem cells or of mesenchymal stem cells or of induced pluripotential stem cells, said composition being obtainable by a method according to any one of claims 1 to 7.

14. Use of composition according to claim 12 or 13 in the preparation of a medicament for the treatment of a hereditary disease or pathological condition related to a genetic defect or a genetic aberration.

15. Use of a composition according to claim 12 or 13 in the preparation of a medicament for the treatment of Pompe disease or a pathological condition related to Pompe disease.

16. A kit comprising means to isolate and/or purify hematopoietic stem cells and a therapeutic gene delivery vehicle.

17. A kit according to claim 16 further comprising instructions to transduce an increased amount of hematopoietic stem cells with a relatively low amount of transducing units per cell of said gene delivery vehicle.

18. A method according to any one of claims 1 to 7, wherein said gene delivery vehicle is provided with means for homologous recombination.

19. A method according to any one of claims 1 to 7, wherein said gene delivery vehicle is provided with means for genome editing.

20. A method according to one of claims 1 to 7, wherein said gene delivery vehicle is provided with means to modify gene expression.

21. A method according to one of claims 1 to 7, wherein said gene delivery vehicle is mutated and provided with means for non-integrating gene expression.