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Gene and Cell-BasedTherapy in Clinical Medicine¶

Chapter 483 | Part 16: Genes, the Environment, and Disease · Parts 15-16 – Genetics, Genomics & Precision Medicine · Chapter 483

Key Clinical Points¶

Gene therapy utilizes nucleic acid sequences (DNA/RNA) as active agents delivered via viral (retroviral, lentiviral, adenoviral, AAV) or nonviral (lipid nanoparticles) vectors.
Two primary strategies for long-term expression: (1) transducing stem cells with integrating vectors (retroviral/lentiviral) so progeny carry the gene; (2) transducing long-lived, postmitotic cells (e.g., neurons, muscle) where even episomal DNA provides stable expression.
CRISPR/Cas9 gene editing uses a Cas9 enzyme and guide RNA to create double-strand breaks (DSB) for precise genome correction or disruption at specific sites.
AAV vectors are nonintegrating, have a ~5 kb packaging capacity, and are ideal for postmitotic tissues like the retina and liver.
Lentiviral vectors are preferred for hematopoietic stem cells due to safer integration patterns compared to retroviral vectors.
CAR-T cell therapies target CD19 (B-cell malignancies) or BCMA (multiple myeloma).
Telomere diseases (e.g., dyskeratosis congenita) manifest as aplastic anemia, cirrhosis, and pulmonary fibrosis; management includes allogeneic transplant and androgens.
Long-term follow-up of 15 years is mandatory for patients receiving integrating vectors to monitor for malignancy.
Key risks include genotoxicity (insertional mutagenesis), immunotoxicity (capsid response), phenotoxicity (overexpression/ectopic expression), and germline transmission.
AAV-mediated therapy can lead to thrombotic microangiopathy due to rapid antibody formation; CAR-T therapy requires management of cytokine release syndrome (CRS) and neurotoxicity.

1. DEFINITION & OVERVIEW¶

• Core Concepts: → Gene Therapy: Active agent is a nucleic acid sequence (DNA or RNA) rather than a protein or small molecule. → Transduction: The process where the vector and donated DNA/RNA enter the target cell and express the transgene. → Delivery Methods: ◦ In vivo: Vector injected directly into the patient. ◦ Ex vivo: Target cells (e.g., hematopoietic, liver, immune) removed, modified in laboratory, then returned to the patient. • Gene Therapy vs. Gene Editing: → Gene Therapy: Includes gene addition, providing a truncated gene with similar activity (e.g., B domain-deleted FVIII), or a gene for an alternative pathway (e.g., utrophin). → Gene Editing: Uses CRISPR/Cas9 (Cas9 enzyme + guide RNA) to create double-strand breaks (DSB) at specific sites for precise correction under endogenous regulatory signals. • Clinical Utility: → Provides treatment for diseases lacking any pharmacologic therapies. → Offers an alternative to complex medical regimens with high nonadherence (e.g., TDT requiring monthly transfusions and iron chelation).

2. EPIDEMIOLOGY¶

• Scope: Transitioned from ultra-rare inherited diseases to more common conditions like sickle cell disease and hemophilia. • Approved Indications: Includes immunodeficiency disorders (ADA-SCID), hematologic malignancies (B-cell lymphoma, multiple myeloma), neurodegenerative disorders (SMA, Metachromatic Leukodystrophy, Adrenoleukodystrophy), retinal dystrophies, hemophilia, muscular dystrophy, and sickle cell disease/β-thalassemia.

3. ETIOLOGY & PATHOPHYSIOLOGY¶

• Mechanism of Action: → Integration of gene transfer techniques with cellular therapies (especially in ex vivo settings). → CAR T-cells have expanded from hematologic malignancies to solid tumors and autoimmune diseases. • Telomere Disease Pathophysiology: → Characterized by telomere erosion and attrition. → Bone Marrow: Leads to aplastic anemia (empty marrow replaced by fat). → Liver: Leads to cirrhosis or nodular regenerative hyperplasia. → Lungs: Leads to pulmonary fibrosis, primarily in subpleural regions.

4. CLINICAL FEATURES¶

• Immunodeficiency Disorders: → Example: X-linked SCID (IL2RG mutation). → Presentation: Infants (first few months) with overwhelming infections and/or failure to thrive. → Outcome: Transduction of autologous CD34+ cells leads to immune reconstitution and growth gains. • Red Cell Disorders: → Conditions: β-thalassemia, sickle cell disease. → Features: Anemia, vaso-occlusive crises, and iron overload. → TDT Standard: Monthly transfusions (Hgb >9 g/dL) + intensive iron chelation. • Neurodegenerative Disorders: → Example: X-linked adrenoleukodystrophy (ALD). → Mechanism: Transduced cells give rise to myeloid cells that cross the blood-brain barrier to become CNS-resident microglia and perivascular macrophages. • Telomere Disease Clinical Features: → Aplastic anemia, cirrhosis, nodular regenerative hyperplasia, and pulmonary fibrosis.

5. DIFFERENTIAL DIAGNOSIS¶

• Bone Marrow Failure Syndromes: → Must distinguish familial clonal hematopoiesis in a long telomere syndrome from other causes of bone marrow failure. → Inherited bone marrow failure syndromes must be differentiated from other causes of cytopenia.

6. INVESTIGATIONS & DIAGNOSIS¶

Genetic Confirmation: Confirm mutations for specific indications (e.g., ADA-SCID, RPE65, SMN1, ARSA, ABCD1, COL7A1, BCL11A).
Hematologic Testing: For sickle cell and β-thalassemia, use hemoglobin electrophoresis and genetic testing.
Telomere Assessment: Diagnosis based on telomere length and clinical features (anemia, cirrhosis, fibrosis).
Monitoring for Adverse Events: → AAV Specific: Monitor for thrombotic microangiopathy and immune responses to capsid. → Genome Editing: Monitor for off-target cleavage and transaminase elevations in liver-directed editing. → CAR-T: Monitor for Cytokine Release Syndrome (CRS) and Neurotoxicity.
Long-term Follow-up Screening (15 years): → New malignancy? → New neurologic/ophthalmologic disorder? → New autoimmune or rheumatologic disorder? → New hematologic disorder?

7. MANAGEMENT & TREATMENT¶

Telomere Disease Management: → Allogeneic hematopoietic stem cell transplant (if donor available). → Lung transplant for pulmonary fibrosis (noted as difficult due to comorbidities). → Liver transplant (successful in several cases). → Pharmacotherapy: Long-term androgens to mitigate telomere attrition. → Precautions: Avoid toxins (metal dust, busulfan, amiodarone), radiation, smoke, and alcohol.
Beta-Thalassemia & Sickle Cell Management: → Lentiviral vector driving expression of βA-T87Q. → Goal: Durable transfusion independence (Hgb ≥9 g/dL and no transfusion for ≥12 months).
CAR-T Therapy Management: → Cytokine Release Syndrome (CRS): Tocilizumab or corticosteroids. → Neurotoxicity: Dexamethasone as early as possible; avoid seizure-threshold-lowering medications. → Immunodeficiency: Prophylaxis for PJP and VZV for ≥1 year; specific vaccination schedule.
Germline Transmission Prevention: → Barrier birth control (noted: AAV vector risk in prostatic fluid, but not in gametes).

8. PROGNOSIS & COMPLICATIONS¶

• Risk Categories (Table 483-3): → Genotoxicity: Insertional mutagenesis or accelerated malignant transformation. → Phenotoxicity: Overexpression or ectopic expression of the transgene. → Immunotoxicity: Harmful immune response to vector or transgene. → Horizontal Transmission: Shedding of infectious vector into environment. → Vertical Transmission: Germline transmission of donated DNA. • Specific Adverse Events (Table 483-4): → Retroviral/Lentiviral: Malignancy, Germline transmission, Immune response to capsid. → AAV: Thrombotic microangiopathy (due to rapid antibody rise). → Genome Editing: Off-target cleavage; Liver-directed transaminase elevation. → CAR-T: CRS, Neurotoxicity, Immunodeficiency, New T-cell malignancy.

9. SPECIAL CONSIDERATIONS¶

• Germline Transmission Risks: → AAV vector risk in prostatic fluid (not gametes). → Action: Barrier birth control until clear of vector DNA. • Immunocompromised States: → CAR-T patients require prophylaxis for PJP and VZV for ≥1 year due to lymphodepleting chemotherapy.

10. KEY PEARLS & CLINICAL TRAPS¶

• Complexity: Gene therapy involves complex interactions between nucleic acids, viral vectors, and cellular responses. → Retroviral/Lentiviral: Requires G phase; 8 kb limit; Extensive immune response (per Table 483-1). → AAV: No G phase required; 5 kb limit; Few immune responses; Nonintegrating. → Lipid Nanoparticles: No G phase; ≥10 kb limit; Transient for RNA. • Clinical Monitoring: Mandatory 15-year follow-up for integrating vectors to monitor for malignancy and other systemic issues.

TABLES & FIGURES¶

• Table 483-1: Characteristics of Commonly Used Gene Delivery Vehicles → Retroviral/Lentiviral: RNA genome, G phase required, 8 kb limit, Extensive immune response, Yes (integrates), Yes (long-term). → Adenoviral: DNA genome, No G phase, 8–30 kb limit, Few immune responses, Poor integration, No long-term. → AAV: DNA genome, No G phase, 5 kb limit, Few immune responses, Poor integration, Yes (long-term). → Lipid Nanoparticles: RNA genome, No G phase, ≥10 kb limit, Immunogenic/Liver target, Can package RNA or DNA, Transient for RNA. • Table 483-2: Currently Approved Gene and Cell Therapy Products → Strimvelis (ADA-SCID); Yescarta (B-cell lymphoma); Zolgensma (SMA); Libmeldy (Metachromatic leukodystrophy); Breyanzi (B-cell lymphoma); Carvykti (Multiple myeloma); Upstaza (AADC deficiency); Hemgenix (Hemophilia B); Beqvez (Hem리면 B); Elevidys (DMD). • Table 483-5: Taking History and Screening → Key questions: Vector type, dose/route, target tissue, transgene nature, and history of adverse events. → Screening for follow-up: Malignancy, neurologic/ophthalmologic, autoimmune/rheumatologic, and hematologic disorders.

Reference Tables¶

TABLE 483-1 Characteristics of Commonly Used Gene Delivery Vehicles FEATURES Genome Cell division requirement Packaging…¶

Harrison's 22e, p.3823

FEATURES	VIRAL BASE		NONVIRAL
	RETROVIRAL/LENTIVIRAL	ADENOVIRAL	AAV	LIPID NANOPARTICLES
Genome	RNA	DNA	DNA	RNA
	G phase 1	No	No
Packaging limitation	8 kb	8–30 kb	5 kb	10 kb or more
		Extensive	Few
Genome integration	Yes	Poor	Poor	May be used to package either RNA or DNA
	Yes	No	Yes
Main advantages	Persistent gene transfer in transduced tissues	Highly effective in transducing various tissues	Elicits few inflammatory responses, nonpathogenic	RNA expressed transiently
	Might induce oncogenesis in some cases; only used ex vivo	Viral capsid elicits strong immune responses	Limited packaging capacity

TABLE 483-2 Currently Approved Gene and Cell Therapy Products in North America and/or Europe PRODUCT Strimvelis® a¶

Harrison's 22e, p.3824

PRODUCT	INDICATION	AGE GROUP	YEAR FIRST APPROVED	WHERE APPROVED	VECTOR	TRANSGENE	TARGET TISSUE
Strimvelis®a	ADA-SCID	Pediatric	2016	Europe	Retroviral	ADA (adenosine deaminase)	Autologous hematopoietic stem cells (HSCs)
	Relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (pediatric); R/R large B-cell lymphoma (adult); third-line follicular lymphoma	Pediatric and adult, different disease indications	2017	United States, Europe, China, Japan	Lentiviral	CAR directed to CD19 with 4-1BB signaling domain
Yescarta® (axicabtagene ciloleucel)	R/R and second-line large B-cell lymphomas; third-line follicular lymphoma	Adult	2017	United States, Europe, Japan	Retroviral	CAR directed to CD19 with CD28 signaling domain	Autologous T cells
	Confirmed biallelic RPE65 mutation–associated retinal dystrophy	Pediatric and adult	2017	United States and Europe	AAV2	RPE65 (retinal pigment epithelial 65 kD protein)
Zolgensma® (onasemnogene abeparvovec)	Spinal muscular atrophy type 1 due to biallelic mutations in the SMN1 gene	Pediatric <2 years of age	2019	United States and Europe	AAV9	SMN1 (survival motor neuron 1)	Spinal motor neurons by single IV infusion
	Transfusion-dependent β thalassemia; sickle cell disease	Adults and pediatric ≥12 years of age	2019	Europe and United States	Lentiviral	βA-T87Q globin gene
Libmeldy®b (aditarsagene autotemcel)	Metachromatic leukodystrophy due to biallelic mutations in the arylsulfatase A gene	Pediatric	2020	Europe, United States	Lentiviral	ARSA (arylsulfatase A)	Autologous HSCs
	R/R mantle cell lymphoma; R/R B-cell acute lymphoblastic leukemia	Adults	2020	United States and Europe	Retroviral	Same molecular construct as axicabtagene
Breyanzi® (lisocabtagene maraleucel)	R/R and second-line large B-cell lymphoma	Adult	2021	United States, Europe, and Japan	Lentiviral	CAR directed at CD19 with 4-1BB signaling domain; CD4 and CD8 T-cell products manufactured and infused separately	Autologous T cells
	Fifth-line treatment for multiple myeloma	Adult	2021	United States and Europe	Lentiviral	CAR directed to B-cell maturation antigen (BCMA); 4-1BB signaling domain
Carvykti® (ciltacabtagene autoleucel)	Fifth-line treatment for multiple myeloma	Adult	2022	United States and Europe	Lentiviral	CAR directed to BCMA with two single-domain antibodies; 4-1BB signaling domain	Autologous T cells
	Early active cerebral adrenoleukodystrophy	Boys age 4–17	2022	Europe and United States	Lentiviral	Adenosine triphosphate– binding cassette, subfamily D, member 1 (ABCD1)
Upstaza® (eladocagene exuparvovec)	Confirmed AADC deficiency with severe phenotype	Children 18 months and older	2022	Europe	AAV2	Human aromatic L-amino acid decarboxylase (AADC)	Cells in putamen via single neurosurgical procedure
	Severe hemophilia A and no history of inhibitors	Adults	2022	Europe and United States	AAV5	cDNA encoding human factor VIII, B domain- deleted, SQ form
Hemgenix® (etranacogene dezaparvovec)	Severe or moderately severe hemophilia B	Adults	2022	Europe and United States	AAV5	cDNA encoding factor IX Padua	Hepatocytes via single IV infusion
	Dystrophic epidermolysis bullosa due to mutations in COL7A1	Age 6 months and older	2023	United States	Herpes simplex viral vector	Collagen type VII alpha 1 chain (COL7A1)
Elevidys® (delandistrogene moxeparvovec)	Duchenne muscular dystrophy	Ages 4–5	2023	United States	AAVrh74	cDNA encoding microdystrophin	Skeletal muscle via single IV infusion
	Sickle cell anemia and β thalassemia	Ages 12 and up	2023	United States and Europe	Gene editing	Inactivates BCL11a in RBCs
Beqvez® (fidanacogene elaparvovec)	Severe or moderately severe hemophilia B	Adults	2024	Canada, United States and Europe	AAVrh74variant	cDNA encoding factor IX Padua	Hepatocytes via single IV infusion

TABLE 483-3 Potential Complications of Gene Therapy Gene silencing—repression of promoter Genotoxicity—complications…¶

Harrison's 22e, p.3825

Gene silencing—repression of promoter
Genotoxicity—complications arising from insertional mutagenesis, or
acceleration of malignant transformation in a cell on the path to oncogenesis
before transduction (i.e., CAR introduced into a premalignant T cell)
Phenotoxicity—complications arising from overexpression or ectopic expression
of the transgene
Immunotoxicity—harmful immune response to either the vector or transgene, or
a harmful immune response of the vector (e.g., CAR T cells)
Risks of horizontal transmission—shedding of infectious vector into environment
Risks of vertical transmission—germline transmission of donated DNA

TABLE 483-4 Adverse Events in Gene Therapy and Gene Editing¶

Harrison's 22e, p.3825

VECTOR OR TREATMENT MODALITY	SYMPTOM OR LABORATORY FINDING	MECHANISM	DOSE DEPENDENCE	MITIGATION STRATEGIES
Retroviral or lentiviral vectors	Malignancya	Insertional mutagenesisa	Yes for retroviral vectors	Less frequent with lentiviral vectors, likely because of differences in integration patterns
	Vector sequences in semen, risk of germline transmission	Based on animal studies, present in prostatic fluid but not in gametes	Yes
	Immune responses directed to capsid, sometimes accompanied by loss of expression	Memory T cells directed to vector capsid in humans, who are natural hosts for wild-type AAV	Yes
	Thrombotic microangiopathy with high-dose systemic infusion	Rapid rise in antibodies to AAV, formation of antigen-antibody complexes, triggering of complement activation	Yes
Ex vivo and in vivo genome editing	Off-target cleavage resulting in unintended gene silencing	Guide RNA lacks requisite specificity	Likely	Preclinical assessment for off-target effects Long-term follow-up of trial participants and patients
	Liver-directed in vivo editing has shown mild and transient transaminase elevations but excellent efficacy at doses studied clinically	Possibly immune responses to bacterial proteins in editing machinery, potentially resulting in loss of edited cells	Yes
CAR-T therapy	Cytokine release syndrome: fever, hypotension, tachycardia, hypoxia, multiorgan failure	Systemic inflammatory response caused by cytokines released by CAR T cells	Possibly	Tocilizumab/corticosteroids
	Neurotoxicity-cerebral edema and encephalopathy	Peripheral immune overactivation, endothelial activation-induced blood-brain barrier dysfunction, CNS inflammation	Possibly	Avoid seizure-threshold-lowering medications in early phase of treatment Treat with dexamethasone as early as possible (use specific management guidelines)
	Immunodeficiency (hypogammaglobulinemia and susceptibility to viral injections)	On-target effect against B cells and/or plasma cells Preparatory lymphodepleting chemotherapy regimen also contributes	No	Prophylaxis for opportunistic infections (PJP, VZV) for at least 1 year; vaccination schedule (specific guidelines)
	New T-cell malignancy	Insertional mutagenesis or potentially chronic activation due to new transgene	No	Report to FDA and manufacturer Consider activation of suicide gene if present in the transgene expression cassette Treat per standard guidelines

TABLE 483-5 Taking History from Patients Who Have Received Gene Therapies or Gene Editing Elements of History for…¶

Harrison's 22e, p.3826

Elements of History for Patients Who Received Gene Therapy (or Have
Participated in Trials)
1. What vector was administered? Is it predominantly integrating (retroviral,
  lentiviral, herpesvirus, or gene editing) or nonintegrating (plasmid, adenoviral,
  adeno-associated viral)?
  2. What were the dose and the route of administration of the vector?
  3. What was the target tissue?
  4. What gene was transferred in? The gene that is defective in the patient’s
  disease? A truncated version? A gene encoding a different protein with
  similar properties? A knockdown approach?
  5. Were there any adverse events noted after gene transfer?
Screening Questions for Long-Term Follow-Up in Gene Transfer
Subjectsa
1. Has a new malignancy been diagnosed? If so, clinicians should contact the
  manufacturer to report the event and obtain instructions on the collection of
  patient samples for testing.
  2. Has a new neurologic/ophthalmologic disorder, or exacerbation of a
  preexisting disorder, been diagnosed?
  3. Has a new autoimmune or rheumatologic disorder been diagnosed?
  4. Has a new hematologic disorder been diagnosed?