Page 1192 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPTEr 85 Gene Therapy for Primary Immune Deficiency Diseases 1157

γ-Retroviral long-terminal repeats are self-inactivated (SIN vectors). Lentiviral
Lentiviral ψ Gene vectors have several potential advantages over γ-RV, including
Foamy viral the ability to more effectively transduce human HSCs in a shorter
α-Retroviral Package as period of ex vivo culture, the capacity to carry longer and more
pseudotyped vector
Administer Gene addition to complex genetic sequences (such as cellular gene enhancers and
marrow stem cells with vector promoters), and less tendency to insert near the 5′ ends of genes
conditioning which may decrease risks for trans-activating expression of the
Collect, isolate adjacent cellular genes. The combination of recombinant cytokines
stem cells used to activate the HSCs to facilitate transduction was advanced
PID ψ Gene as factors were identified that acted on the earliest HSCs, including
patient Transplant flt-3 ligand and thrombopoietin, combined with c-kit ligand.
gene-modified
stem cells Role of Cytoreductive Conditioning to
Gene correction of stem cells
with site-specific nuclease Facilitate Engraftment
and homologous donor Initial trials of gene therapy for PID did not administer pre-
FIG 85.2 Autologous Transplantation of Gene-Corrected transplant cytoreductive conditioning, due to potential risks of
Hematopoietic Stem Cells (HSC). Gene addition may be per- chemotherapy or radiation with unknown prospects of benefit
formed using Retroviridae-derived vectors (from gamma-retro- from the gene transfer procedure. It is well-known from multiple
viruses, lentiviruses, foamy viruses or alpha-retroviruses) to transplant studies in mice and large animal models that there
transfer a normal copy of a relevant gene into HSCs collected is minimal if any engraftment of autologous HSCs when given
and isolated from a primary immune deficiency (PID) patient. without prior conditioning, unless extraordinary numbers of
The gene-containing vector is packaged in a suitable envelope cells (e.g., 30–50x higher than standard cell dose/kg) are given;
(pseudotyped) for gene addition to human HSC. Alternatively, even mega-dose transplants without conditioning lead to only
the HSC may be gene-corrected using site-specific endonucleases low levels of engraftment (e.g., 1%), although this may be
1
to augment homologous recombination–directed gene correction. persistent. While there was initial reluctance to use conditioning
The patient may receive marrow cytoreductive conditioning to in gene therapy trials when there had not previously been efficacy,
enhance engraftment after transplant of the gene-modified stem now with the clear-cut benefits that may be obtained from gene
cells. therapy, the necessity to use conditioning for autologous gene
therapy HSCT (which is less than that needed for allogeneic
HSCT) is becoming recognized as the standard. The amount
and types of chemotherapy drugs used for conditioning has
varied, depending on the disease setting. For SCID, where a
relatively low level of gene-corrected HSCs may support immune
reconstitution, reduced-intensity conditioning (RIC), such as
THEraPEUTIC PrINCIPLES relatively low dose busulfan alone (e.g. 4–6 mg/kg), may be
Steps for Gene Transfer to Hematopoietic Stem sufficient. For other disorders where less of a selective advantage
Cells for Clinical Transplantation may exist for the gene-corrected cells, a higher level of engraftment
of modified HSC may be needed, and thus stronger conditioning
Package as Pseudotyped Vector regimens have been used, reaching levels for myeloablative
Transfect packaging cell with vector plasmid and virion/envelope conditioning (MAC) (e.g., busulfan, 12–16 mg/kg). For the WAS
plasmids. where it may be necessary to ablate the pre-transplant immunity
Collect released vector from cell culture medium (DNAse). to reduce risks for post-transplant autoimmunity, immune-
Purify and concentrate vector (e.g., ion-exchange chromatography, suppressive drugs (e.g., fludarabine, rituximab) have been added
tangential flow filtration).
Aliquot. Certify. Release. to conditioning regimens. These combined iterative approaches
to improving gene therapy have led to the current state, where
Transduce Stem Cells With Vector clinical benefits are being routinely achieved, as detailed below
+
Isolate CD34 stem/progenitor cells from clinical source (bone marrow, for each disorder.
mobilized peripheral blood, cord blood). New methods are being explored to “make space” in the
+
Grow CD34 cells in serum-free medium plus recombinant cytokines marrow using alternatives to chemotherapy and radiation, such
(e.g., ckit ligand, flt-3 ligand, thrombopoietin). as monoclonal antibodies to markers present on HSCs. These
Add vector to cells and culture for transduction. are moving toward clinical assessments and may in the future
Link vector sequences covalently into stem cell’s chromosomal DNA.
Formulate and characterize cell product for release. facilitate engraftment with lower short-term and long-term risks
than with chemotherapeutic agents.
administer Marrow Cytoreductive Conditioning
May deliver single or combination chemotherapeutic agents or monoclonal Adenosine Deaminase (ADA)-Deficient Severe
antibodies to “make space” in marrow. Combined Immune Deficiency (SCID; Chapter 35)
The first clinical trial of gene therapy for an inherited disorder
Transplant Gene-Modified Stem Cells (other than a premature attempt at gene therapy for beta-
Infuse gene-modified cell product intravenously. thalassemia) was directed against adenosine deaminase (ADA)-
Stem cells engraft and transmit transgene to all progeny blood cells. deficient Severe Combined Immune Deficiency (SCID).
Transgene produces necessary gene product to correct genetic ADA-SCID was the first form of human SCID for which the
deficiency.
responsible gene was identified and cloned, allowing gene therapy